Exome Sequencing Reveals Primary Immunodeficiencies in Children with Community-Acquired Pseudomonas aeruginosa Sepsis

One out of three pediatric sepsis deaths in high income countries occur in previously healthy children. Primary immunodeficiencies (PIDs) have been postulated to underlie fulminant sepsis, but this concept remains to be confirmed in clinical practice. Pseudomonas aeruginosa (P. aeruginosa) is a common bacterium mostly associated with health care-related infections in immunocompromised individuals. However, in rare cases, it can cause sepsis in previously healthy children. We used exome sequencing and bioinformatic analysis to systematically search for genetic factors underpinning severe P. aeruginosa infection in the pediatric population. We collected blood samples from 11 previously healthy children, with no family history of immunodeficiency, who presented with severe sepsis due to community-acquired P. aeruginosa bacteremia. Genomic DNA was extracted from blood or tissue samples obtained intravitam or postmortem. We obtained high-coverage exome sequencing data and searched for rare loss-of-function variants. After rigorous filtrations, 12 potentially causal variants were identified. Two out of eight (25%) fatal cases were found to carry novel pathogenic variants in PID genes, including BTK and DNMT3B. This study demonstrates that exome sequencing allows to identify rare, deleterious human genetic variants responsible for fulminant sepsis in apparently healthy children. Diagnosing PIDs in such patients is of high relevance to survivors and affected families. We propose that unusually severe and fatal sepsis cases in previously healthy children should be considered for exome/genome sequencing to search for underlying PIDs.

inTrODUcTiOn Despite worldwide reduction in childhood mortality, sepsis remains one of the leading causes of childhood deaths. Importantly, 35-50% of pediatric sepsis deaths occur in previously healthy children (1)(2)(3). These children often develop non-specific symptoms suggestive of a viral respiratory infection, followed by sudden deterioration and rapid progression to shock and multisystem organ failure. In addition to pathogen virulence factors, such as streptococcal Toxic Shock Toxin (4), rare genetic variants causing a primary immunodeficiency (PID) may underlie fulminant sepsis. Such variants have high effect sizes and are usually kept at low frequencies in the population due to purifying selection (5,6). Previous studies searching for PID in children with bacterial sepsis were limited to conventional immunological testing (5). Using high-throughput sequencing studies on individuals with extreme phenotypes, who are most likely to be informative from a genetic point of view, has been demonstrated to be very powerful in Mendelian diseases but has not been reported in patient cohorts with invasive bacterial infections (6).
Pseudomonas aeruginosa (P. aeruginosa) is an aerobic Gramnegative bacterium commonly found in environment. This opportunistic pathogen causes invasive infections in immunosuppressed and hospitalized patients and represents a major cause of health care-related infections. In contrast, sepsis due to community-acquired P. aeruginosa is extremely rare in apparently healthy children, and carries very high mortality (7,8), and may be the first manifestation of an underlying PID. Indeed, a few case reports have already described the identification of PIDs in children with P. aeruginosa sepsis (9,10). Here, we used exome sequencing and bioinformatic analysis to identify genetic variants conferring extreme susceptibility to P. aeruginosa in a cohort of 11 previously healthy children.

Patients
Children below 60 months with community-acquired blood-culture positive P. aeruginosa sepsis were eligible. Children with any comorbidities (prematurity, congenital malformations, previous surgery, immunosuppression, known immunodeficiency, and chronic diseases) and children who had been exposed to broadspectrum intravenous antibiotics were excluded. This study was carried out in accordance with the recommendations of the ethics committees of participating centers (Kantonale Ethikkommission Bern, KEK Ref Nr 029/11, Bern, Switzerland; Mater Health Services HREC13/MHS/4, Brisbane, QLD, Australia). Parents/ guardians of all included patients gave written informed consent in accordance with the Declaration of Helsinki. Patients were recruited prospectively and retrospectively in hospital databases and infectious diseases networks. Whenever possible, DNA was also collected from the parents.

Variant calling
We used genome analysis toolkit (GATK) (13, 14) version 3.1-1 to call single nucleotide variants (SNVs) and small insertion and deletions (indels) from duplicate-marked bam files. We used HaplotypeCaller for multi-sample variant calling on all samples followed by GATK best practice to call the variants and included only variants that were flagged as PASS by GATK in subsequent analysis.

Variant effect Prediction
We used SnpEff (15) version 4.1B to predict the functional impact of variants. As variants can have several predicted effects, we only considered the most severe effect for each variant. The effects in decreasing order of severity are frameshift and in-frame for indels and stop-gain, splice site-disrupting, non-synonymous, synonymous, intronic, UTR, non-coding exon, and intergenic for SNVs. Variants were annotated as putative loss-of-function (LoF) if they were stop-gain or splice site-disrupting SNVs or frameshift indels mapping to the first 95% of coding region, or larger deletions removing either the first exon or more than 50% of the protein-coding sequence of the affected transcript.

identification of Potentially causal Variants in Parent-child Trios
We restricted analyses to non-synonymous and LoF exonic variants with MAF <1% in ESP, 1KG, UK10K, ExAC, and in-house control exomes. We analyzed each family separately assuming autosomal recessive, autosomal dominant, and X-linked recessive inheritance models. Only variants with >10× coverage in both the parents and the offspring were included in the de novo analysis. We assumed full penetrance for the potentially causal variants and given the fatality of the phenotype did not consider a mutation as potentially causal if it was present in the parents, any of the abovementioned databases or in the in-house control exomes in the same zygosity form (i.e., heterozygous or homozygous) as in our patients.

identification of Potentially causal Variants in individual Patients
We restricted analyses to non-synonymous and LoF exonic variants with MAF <1% in ESP, 1KG, UK10K, ExAC, and inhouse control exomes. We analyzed each individual separately assuming autosomal recessive and X-linked recessive inheritance models. We assumed full penetrance for the potentially causal variants and given the fatality of the phenotype did not consider a mutation as potentially causal if it was present in any of the abovementioned databases or in the in-house control exomes in the same zygosity form (i.e., heterozygous or homozygous) as in our patients.

Targeted search in Primary immunodeficiency genes
We ran a targeted search for rare (MAF <1%), non-synonymous, and LoF variants in a list of 252 known PID genes. This list includes known genes in 50 PID syndromes compiled by The International Union of Immunological Societies (IUIS) Expert Committee in 2015 (20) and 3 newly published PID genes since the latest release of IUIS till April 2016 ( Table 1).

gene-annotation enrichment analysis
We used the database for annotation, visualization, and integrated discovery (DAVID) (24,25) v6.7 with default options and highest classification stringency for functional annotation clustering of PID genes carrying rare, non-synonymous variants. We used hypergeometric test for assessing the significance of gene enrichment results.

c9 reconstitution experiment
Serum of a patient heterozygous for a complement C9 variant was diluted with GVB ++ in a series starting at 1:10 to 1:640 in 1:2 steps. The same was done with a serum pool as a control. This pool consisted of serum from over 300 healthy donors. Forty microliters of these dilutions were pipetted in duplo in a roundbottom 96-wells plate. To these wells, 40 μl of GVB ++ was added to the samples and the blanc, to the positive control 40 μl of 1.7% saponin was added (100% lysis). Ten microliters of purified C9 (Quidel) or GVB ++ were added (end conc 50 μg/ml). Next, in all wells, 150 μl of EA's were added. EA's are sheep erythrocytes (Hatunalab, Sweden) coated with an optimal dose of Amboceptor (Rabbit-anti-Sheep erythrocyte; Dade Behring) brought to a concentration of 0.25 × 10 8 cells/ml. This was incubated for 1 h at 37°C while agitating. After incubation, the plate was centrifuged for 5 min at 2000 rpm. Fifty microliters of each supernatant were pipetted to a flat-bottom 96-wells plate and diluted with 150 μl GVB ++ . Extinction was measured at 415 nm using a spectrophotometer. The percentage of lysis was calculated as follows: GVB ++ buffer consisted of 2 mM 5,5 di-ethylbarbituurzuur (Genfarma), 1.15 mM Na 5,5 di-ethylbarbituraat (Bufa), 96 mM NaCl (Merck), 0.5 mM CaCl (Merck), and 0.18 mM MgCl2 (Merck).

resUlTs clinical Presentation
We identified 11 previously healthy children with communityacquired P. aeruginosa bacteremia from whom DNA could be obtained. Child-parent DNA trios were available in seven affected families. The presenting age ranged from 6 months to 4 years, and 7/11 (64%) of patients were male ( Table 2). All patients had never been admitted to hospital or exposed to intravenous antibiotics prior to presenting with P. aeruginosa sepsis. Nine patients had developed respiratory symptoms within 72 h of leading to hospital admission, one presented with Ecthyma gangrenosum (Figure 1), and eight (73%) died. Three children died in the emergency department shortly after presentation, and all deaths occurred within 48 h of hospital admission. Postmortem examination reports were available in seven cases. In four, P. aeruginosa grew in high quantities from nasal and oral swabs, tracheal secretions, and lungs. Viral co-infections were found in five deceased patients, including human herpes virus-6, parainfluenza virus-3, human metapneumovirus, respiratory syncytial virus, and varicella virus. One surviving patient presented with acute abdomen, and P. aeruginosa bacteremia was thought to result from intraabdominal perforation. Another surviving patient was diagnosed with urosepsis and P. aeruginosa grew in urine and blood. The third surviving patient presented with recurrent parainfectious neutropenia. Six patients received aminoglycosides and/or anti-Pseudomonas beta-lactam antibiotics during sepsis, including all three survivors. exome sequencing and short read alignment FigUre 1 | ecthyma gangrenosum, a highly suggestive rapidly progressive purpuric skin lesion seen in a minority of children with P. aeruginosa bacteremia, is shown in one of the study patients (with permission from parents).

Variant analysis
Thirty-eight rare, non-synonymous coding variants (MAF <1%) were observed in the 11 patients after testing for different inheritance models ( Table 3), among which 12 were never seen in the same zygosity form (heterozygous or homozygous) in publicly available databases ( Table 4). In addition to the above 38 variants, we performed a targeted search for rare, non-synonymous coding variants in 252 previously known PID genes and found 76 variants in 61 genes ( Table 5). Functional classification of these 61 genes using DAVID highlighted the complement pathway as the most enriched cluster (DAVID enrichment score: 15.73). In total, 14 of the 28 complement pathway genes present in the list of 252 known PID genes carried at least one rare, non-synonymous coding variant in our cohort (hypergeometric probability: p(x > 14) = 0.003).
In one patient (patient S4) who died of fulminant septic shock, we identified a novel single-base insertion on the X chromosome (C > CT, chromosome 20, position 100617191) leading to a frameshift in the Bruton agammaglobulinemia tyrosine kinase (BTK) gene. Other than a middle ear infection during infancy, the previous history, growth, development, and vaccination history of the patient had been unremarkable. Taqman genotyping and clinical genetic testing confirmed presence of mutation in the deceased patient and revealed a de novo occurrence in the mother, while other family members were healthy. BTK plays a crucial role in B-cell development. Postmortem serological testing confirmed the absence of immunoglobulins, consistent with BTK LoF: IgG < 1.3 g/l (4.22-11.9); IgA < 0.2 g/l (0.2-1.58); IgM < 0.2g/l (0.48-1.9). A novel homozygous deletion spanning 6 bp on chromosome 20 (ACTCGAG > A, X chromosome, position 31393171) was observed in a 2-year-old boy with consanguineous parents (patient S13), leading to an in-frame deletion in a conserved region of the catalytic domain of the DNMT3B gene. Previously described missense mutations in the same protein domain are known to cause immunodeficiency-centromeric instability-facial anomalies (ICF) syndrome (26), a rare disease characterized by variable immunodeficiency and recurrent infections with mild facial abnormalities. The fatal septicemia was preceded by uncomplicated varicella, and the patient had a history of recurrent mild respiratory and upper airway infections that had been attributed to poor health conditions. The patient had normal T and B cell counts but reduced immunoglobulin levels, consistent with ICF syndrome: IgG 0.16 g/l (4.22-11.9); IgA <0.06 g/l (0.2-1.58); IgM 0.05 g/l (0.48-1.9).
We also observed a rare, known (MAF = 0.001 in ExAC) heterozygous stop-gain variant in the complement C9, on chromosome 5 (G > T, chromosome 5, position 39342214) in a male patient (patient S15) with severe Pseudomonas septicemia and recurrent parainfectious neutropenia. The patient survived without sequelae. Complement reconstitution assay with classical or terminal pathway proteins (C1-C9) showed normal lytic activity  (Figure 2), suggesting that this variant is unlikely to be causal to increased susceptibility to P. aeruginosa.

DiscUssiOn
We studied P. aeruginosa sepsis in previously healthy children as a model disease defining an extreme phenotype of fulminant sepsis. Fulminant sepsis in children is associated with high mortality, and the majority of deaths occur within hours of presentation (27). The disease burden is highest in children under 5 years of age (3,7). We hypothesized that rare genetic variants with high effect sizes could explain life-threatening susceptibility to P. aeruginosa in children without known risk factors or comorbidities. Mendelian disorders of immunity have previously been shown to result in phenotypes with unusual susceptibility to bacterial infection, such as pseudomonal and pneumococcal infections in patients with IRAK-4 deficiency (10,28). We used exome sequencing to explore the genetic cause of susceptibility to P. aeruginosa in a carefully selected group of previously healthy children with no familial history of immunodeficiency, who developed community-acquired P. aeruginosa septicemia. By systematic search for fully penetrant, rare, non-synonymous, and LoF variants in exonic regions, we identified 12 potentially causal variants including two novel variants in known PID genes: X-linked agammaglobulinemia due to a novel BTK mutation and ICF immunodeficiency syndrome due to a novel DNMT3B mutation. In addition, we performed a targeted search for rare, non-synonymous, and LoF variants in known PID genes and observed an enrichment of such variants among genes that are part of the complement pathway. One patient carried a heterozygous, stop-gain variant in the C9 gene, which has been associated with recurrent meningitis (29,30). However, functional testing of patient samples showed normal lytic activity, demonstrating the need for strict validation of potentially causal mutations, even in genes with plausible biological links to the study phenotype.
Variants conferring life-threatening susceptibility to common infections in children below reproductive age are subjected to strong purifying selection and will therefore only be found at very low frequencies (31)(32)(33). Out of the 12 potentially causal variants described in this study, 11 were novel and 1 was a hemizygous variant present in 1 individual (in heterozygous form) in ExAC. Previous case reports describing P. aeruginosa sepsis in children with known PIDs, such as Wiskott-Aldrich syndrome, X-linked agammaglobulinemia, cyclic neutropenia, and IRAK-4/MyD-88 deficiency (8,10,22,34), were based on conventional, candidate-gene-driven immunological testing. Ecthyma gangrenosum is present in a minority of patients and has been described as the presenting sign of an underlying PID, such as cyclic neutropenia, chronic granulomatous disease, or hypogammaglobulinemia (35)(36)(37). The range of PID types identified in these previous reports indicates that multiple distinct genetic defects have to be considered when investigating such clinical presentations. Importantly, classic PID screening may fail to identify new mutations in known PID genes (38) and will miss causal mutations in genes without an established link with PIDs. Failure to diagnose rare underlying PID in a child presenting with sepsis may result in potentially devastating consequences for survivors, undiagnosed siblings, and their families. High-throughput sequencing, in particular exome sequencing, has proved highly successful in the clinical diagnosis of suspected monogenic conditions in the pediatric population (39)(40)(41). This was recently illustrated by Record et al., who used exome sequencing in a girl with P. aeruginosa sepsis and found a homozygous LoF mutation in MAP3K9 (MKL1), a gene that was not known to cause PID (22).
A strength of the present cohort is that the case definition required significant growth of community-acquired P. aeruginosa in blood culture in previously healthy children presenting with signs and symptoms of sepsis, thereby constituting an extreme phenotype. The majority of children included in this study presented with fulminant sepsis and septic shock due to Pseudomonas sepsis. In the cases where postmortem examinations were performed, extensive growth of P. aeruginosa was found in several tissues, suggesting overwhelming bacterial infection. In agreement with other studies, we observed that several sepsis patients had viral co-infections (3). The role of viral  co-infections in the pathogenesis of childhood bacterial sepsis is poorly understood and may include facilitated bacterial invasion due to respiratory epithelial disruption and increased host susceptibility during viremia (42). Immunological investigations in children presenting with fulminant sepsis can be extremely challenging, as severe leukopenia, coagulopathy, multi-organ failure, and fluid resuscitation often lead to severe alterations of cell counts, immunoglobulin levels, and complement levels.
In addition, a proportion of patients die prior admission to an intensive care unit, or present with out-of-hospital cardiac arrest or sudden infant death syndrome (43). To date, there are no widely accepted guidelines to inform pediatricians, emergency and intensive care physicians, and pathologists about indications for specific immunological investigations in previously healthy children presenting with fulminant or fatal sepsis (44). Given the high fatality in our cohort, with several cases recruited considerable time after death of the patient, our study highlights the value of performing exome sequencing in this population using blood or tissue containing DNA. However, the lack of viable host cells in deceased patients may represent a major limitation toward functional validation of potentially causal variants. Elucidating the role of these variants in novel genes that may confer a PID phenotype will therefore require independent validation in other patients or cohorts. While it is well known that PIDs can be responsible for severe bacterial infections, little is known about the proportion of children with invasive infections suffering from PID (5). In our cohort, defects in known PID genes were found in 25% of fatal cases. This is comparable to a recent study using exome sequencing in 50 patients with common variable immunodeficiency where exome sequencing identified disease-causing mutations in 30% of cases (45). While non-genetic factors, including variation in pathogen virulence and secondary neutropenia, may be responsible for the remaining cases, we cannot rule out that mutations in genes not previously associated with PID are at least partially involved. Sequencing of additional family members, sequencing of more cases with the same phenotype, and functional characterization of new candidate genes and variants will be required to identify such genetic factors. Furthermore, whole-genome sequencing is needed to explore the non-coding variants, large structural variants, and also exonic variants that are not be well-covered using exome sequencing.
In conclusion, this study provides proof of concept that exome sequencing allows the identification of rare genetic variants responsible for fulminant sepsis in children in whom immunodeficiency had not been previously suspected. Given the decreasing cost of exome and genome sequencing (39,40,46), we propose considering host DNA sequencing as a possible diagnostic procedure for apparently healthy children presenting with unusually severe or fatal sepsis.