Whole-exome sequencing metrics
The whole exomes of the seven pediatric SCLS patients and their unaffected first-degree relatives (in total 26 samples, Supplemental Figure 1A, http://links.lww.com/SHK/A819) were sequenced to a mean depth of 76.5 independent reads per targeted base across all the samples. Across all samples, 97.1% of all targeted bases had more than eight independent reads. The nine adult SCLS patients were sequenced previously to a mean depth of 35.8 reads. The mean of 83.7% of targeted bases, across the nine samples, had more than 8X coverage. This depth provides enough-confidence calling of homozygous and heterozygous variants across the exomes (Supplementary Tables 1 and 2, http://links.lww.com/SHK/A819).
Principal component analysis (PCA) from WES genotypes showed that the majority of patients were of European ancestry with one patient of each African ancestry and Mexican ancestry (Supplemental Figure 2B, http://links.lww.com/SHK/A819). This result agrees with the patients’ self-identified ethnicities. The minor allele frequency used to analyze each sample was subsequently adjusted to account for ethnicity.
De novo variant analysis
No SCLS patients in either cohort had any family history of the disease, rendering traditional gene mapping strategies in extended families impossible. Therefore, we analyzed parent–child trios, a validated approach for gene discovery, with a focus on rare recessive and de novo variants (20). There were five such trios available in the current study. We identified only one de novo coding variant (c.A5765G, p.Q1922R) in the DNAH11 gene from one family (Table 3). This variant was novel as it was not identified in 123,126 exomes in the gnomAD database, and the encoded amino acid was highly conserved across human evolution. DNAH11 encodes Dynein Axonemal Heavy Polypeptide 11, a protein involved in microtubule-dependent motor ATPase movement of respiratory cilia; mutations have been associated with primary ciliary dyskinesia (21). However, DNAH11 is also expressed in endothelial cell lines, and microtubule function has essential roles in vascular barrier function (22).
Recessive inheritance analysis
Next, we searched for rare homozygous or compound heterozygous disease-causing nucleotide variants that may follow recessive inheritance. Among our cohort, there were seven patients with first-degree relatives available for sequence comparisons. Parental study allows for ready determination of whether any particular allele is inherited from the maternal or paternal side. Frequently, sequence analysis of only one parent may permit such a determination when sequence from the other parent is not available. If only one allele is present in the available parent, the other allele either must have been inherited from the other parent or occurred de novo. Using this approach, we could distinguish between compound heterozygous and biallelic variants if they are inherited in trans. If the inheritance of the biallelic variants is not distinguishable due to lack of parent genetic testing, and they were present in the 1,000 genomes dataset with strong linkage disequilibrium (D’ > 0.9) demonstrated within a population, they were excluded. Because a recombination between them is unlikely to be happened in the genome. We identified multiple compound heterozygous (Table 4), biallelic heterozygous (Supplementary Table 3, http://links.lww.com/SHK/A819), and homozygous or X-linked hemizygous (Supplementary Table 4, http://links.lww.com/SHK/A819) variants that warranted investigation as possibly contributing to the pathogenesis of SCLS. We included variants with a relatively high allele frequency (≤0.5% subpopulation frequency) and applied liberal criteria to assess the impact of nucleotide variations on protein function and disease-causing potential (combined annotation dependent depletion [CADD] score ≥15) to maximize the possibility for discovery, which may identify a few false positive target genes. Despite these relatively low thresholds, we did not identify rare gene variants common to all patients, nor were there mutations in genes that were common to multiple patients but containing distinct mutations in individual patients.
Rare heterozygous variant analysis
Finally, we sought rare heterozygous variants in the nine unrelated adult subjects. We used a dominant inheritance model to identify novel or highly rare alleles (MAF≤0.01%) and focused on variations predicted to be highly deleterious (CADD score ≥20) or lead protein-truncation. We also used gene-based collapsing analysis of these variants (15) for discovery of genes containing potentially pathogenic mutations. Collapsing analysis identifies variations of potential pathologic importance in all relatives, thereby producing a large list of potentially implicated genes, which much is analyzed in the appropriate clinical and biological pathway context. To maximize the impact of the identified gene targets, we performed extensive quality control and screened each aligned read to avoid any false positive variants that could give spurious findings. The analysis of very rare heterozygous protein-truncating variants yielded 46 genes of interest, which are listed in Supplementary Table 5 (http://links.lww.com/SHK/A819). No common genes or metabolic pathways with rare protein-truncating heterozygous variants were identified. The gene-based analyses, which collapse all rare loss of function (LOF) and damaging variants on a gene level, did not provide any statistically significant enrichment for a disease-associated gene. This analysis identified 10 genes of potential interest and are listed in Supplementary Table 6 (http://links.lww.com/SHK/A819).
In this report, we detail the clinical characteristics and WES genetic analysis from pediatric and adult SCLS cohorts. This investigation represents the most comprehensive genetic analysis to date exploring a potential underlying genetic etiology of SCLS. Although no common genes or pathways were detected across either the adult or pediatric cohorts, in-depth analysis identified multiple single nucleotide variations that may have relevance to individual patients. These results suggest that the SCLS phenotype is not explained by germline exomic mutations either in a single gene or known pathway. Our results do not definitively exclude genetic variation as a contributing etiology to SCLS. Other mechanisms of genetic influence may be considered, such as locus heterogeneity, when mutations in several different genes can lead to a similar clinical phenotype. In complex biological processes such as vascular permeability, a large number of genes may interact to produce a common phenotype. This hypothesis could be tested by further basic science research into rare variants in various genes that were identified in individual patients in our cohort. Furthermore, SCLS could also be a result of genetic changes in noncoding regions, such as variants in intronic sequences or epigenetic modifications. Evaluation of this possibility would require additional approaches such as whole-genome sequencing or DNA methylation analyses. A third alternative is that SCLS could result from complex interactions of variants of two or more genes. Our current study does not provide support for or against these more complex genetic mechanisms of genetic regulation in SCLS.
Nongenetic etiologies for SCLS must also be considered. For example, the paraprotein associated with MGUS may act through an as-yet undescribed mechanism to induce leak in adult patients, although this would not explain pediatric patients with SCLS. Other possibilities include occult infection or subclinical insult that alters the systemic permeability or inflammatory response. Possibilities include viral infections that alter VEGF, angiopoietin-2, tumor necrosis factor or IL-2 signaling (8), or an unknown process that promotes endothelial apoptosis (23). Evidence for such competing hypothesis is scarce; however, our results suggest that investigations directed at novel hypotheses may be worthwhile.
In pursuing this study, we also show the feasibility and applicability of WES in investigating the etiology of disease with presume genetic contribution in the ICU setting. Historically, when caregivers suspected a genetic component to their patient's illness, several obstacles delay the rapid identification of monogenic defects contributing to disease. Specimens may be difficult to obtain from pediatric or acutely ill adult patients. In addition, even with timely acquisition and analysis of sequencing, interpretation of these tests may be attempted by researchers with little understanding of the clinical syndrome in question. Critical care clinician researchers and their trainees, on the contrary, are well positioned to combine clinical knowledge and data with genetic analysis which may lead to meaningful discoveries (24). Such innovation is already underway in neonatal intensive care units where genomic technologies are being increasingly used (25). As we have demonstrated here, such patient-focused research efforts have the potential to improve our understanding of how specific genetic abnormalities contribute to disease pathogenesis. Although no unifying genetic etiology of SCLS was identified, we uncovered a number of noteworthy findings that may impact the care of current and future SCLS patients.
The clinical characteristics reported in these cohorts are consistent with those previously reported (4, 26). Although a consistent feature of pediatric SCLS is its linkage to antecedent infections, infectious triggers are more variably (50%–75%) identified in adults (7). As previously reported, MGUS associated with adult SCLS patients was not observed in our pediatric cohort (26). In addition, the overall number of severe episodes and mortality in both cohorts are lower than previously reported, possibly due to the recent advance of immunoglobulin therapy to prevent serious episodes (12, 27). The high mortality in those presenting with undiagnosed disease supports his theory. In short, the adults and children studied are highly clinically representative of SCLS cases reported in the medical literature and thus an excellent cohort for WES analysis.
De novo variant analysis
Sequencing of trios is a powerful tool to detect the presence nucleotide variants present in probands absent in the parents (Table 3). Such variations are often highly significant for disease processes, although there are natural variations in genomic DNA that may have no impact on protein functions. Our analysis found only one individual with a single de novo variation in the gene DNAH11, which encodes dynein axonemal heavy chain 11, a protein component of cilia. It is mainly expressed in ciliated cells in the fallopian tubes and respiratory tract, as well as in parathyroid and adrenal glands (28). Mutations of the gene are associated with Ciliary Dyskinesia and situs inversus, but there are no reports of vascular leak-related symptoms in these disorders (29). Although DNAH11 variants are associated with lethal respiratory and cardiac lesions in mice (30), there is no mechanistically plausible link of DNAH11 to SCLS. WES may detect small numbers (less than three) of normal de novo coding variants in any given patient, raising the likelihood that this finding is simply a rare normal variant and unlikely to explain the patient's SCLS.
Recessive and compound heterozygous inheritance analysis
Inheritance of two defective alleles results in autosomal recessive disorders. We identified 21 genes with rare single nucleotide variations that were likely to adversely affect protein function as assessed by in silico prediction algorithms. A single adult patient had biallelic mutations in DOCK6, although compound heterozygous status is uncertain due to lack of family inheritance information. DOCK6 encodes Zir1, a GEF for the RhoGTPases Cdc42 and Rac1, both of which are known to enhance vascular barrier function through stabilization of tight junctions (30). As a GEF, Zir1 would function as a molecular “on” switch for Cdc42 and Rac1 (Fig. 1). A defect in the “on” switch for a RhoGTPase that acts to fortify vascular barriers may very well produce the signs and symptoms of excessive vascular leak observed in SCLS. Some mutations in DOCK6 in humans have been associated with congenital finger and toe abnormalities in Adams-Oliver Syndrome (31). Finally, although it is widely expressed in multiple tissues, DOCK6 exhibits highest expression levels in endothelial cell lines, making it a potentially attractive target. We are currently planning further studies into its role in endothelial permeability pathways.
Multiple other genes of potential interest for SCLS pathogenesis were identified as noted in Table 4 and Supplemental Table 3 (http://links.lww.com/SHK/A819). Although numerous, these results must be interpreted in the specific clinical setting, highlighting the importance of pediatric critical care providers engaging in this research. Genes that are involved in cell signaling include PIGT, SH3KBP1, MST1R and PELP1. PIGT encodes a glycosylphosphatidylinositol (GPI)-anchor that cleaves membrane bound molecules. Mutations in this gene have been associated with developmental delay (32). Other genes identified include CACNAC1C (a voltage gated calcium channel), AIFM3 (a mitochondrial apoptosis sensor), DNAH17 (a cilia component), F11 (coagulation factor XI), PASD1, ZNF407 (both nuclear binding proteins), PAGE3, SRP68, NPC1L1, TECPR1 (all protein binding genes), TANC2, SRPX, PAPLN, and PXDN (all matrix proteins). To our knowledge, none of these gene products has been implicated in endothelial dysfunction. This provides us with the unique opportunity to potentially uncover dysregulated expression patterns and/or unexpected functions related to vascular barriers in one or more of these candidates. Of note, although the functional significance remains unclear, CACNAC1C was identified as enriched in SCLS patients using genome wide association studies (13).
Rare heterozygous protein-truncating and deleterious missense variant analysis
Due to the extremely low minor allele frequencies, detection of individual rare causal variants is difficult. To increase the power for detection, rare variant burden testing usually requires aggregation of the heterozygous rare LOF and deleterious variants (CADD ≥20) into a gene or a pathway as a unit for statistically significant enrichment. We did not detect any common pathways shared by rare protein-truncating, deleterious variants after adjustment by genetic constraint or size of the gene; however, we did identify individual genes of interest (Supplementary Table 4, http://links.lww.com/SHK/A819). A good example is JUP, the gene encoding plakoglobin. Expressed at endothelial cell junctions, plakoglobin prevents barrier dysfunction induced by permeability mediators such as vascular endothelial growth factor A (VEGFA) by interacting with junctional proteins such as the receptor tyrosine phosphatase, VE-PTP, and VE-cadherin (33). As serum levels of VEGFA are increased in patients with SCLS specifically around disease flares, impaired plakoglobin function could induce and/or magnify the clinical symptoms of vascular leakage in these patients (8, 34). JUP is also highly expressed in the cytoplasm and membrane of epithelial cells, especially in the esophagus and skin, neither of which are specifically more affected in SCLS. Mutations of JUP reported thus far have resulted in primarily in dermatologic disease, although cardiac disease has also been described (35).
Our study has several limitations. Because SCLS is a highly rare disease, we had limited patient enrollment. We sequenced eight children and nine adults with SCLS, and thus our study is underpowered due to the extreme rarity of the disease. Increased patient enrollment will greatly assist in discovery, especially for rare heterozygous loss of function variants. Three of our pediatric patients and all nine of our adult patients did not have both parents sequenced, limiting our de novo analysis.
We were unable to identify variants in a gene or pathway common to all patients via WES. However, our previous work with a single SCLS patient (9) suggests that genetic defects may still contribute to exaggerated vascular permeability signaling with clinical consequences that match the signs and symptoms of SCLS. Given the lack of a single identified germline exomic etiology, other possible mechanisms include somatic (nongermline) mutations of low frequency that are below the threshold of detection by WES, mutations in noncoding DNA regions, unknown epigenetic changes, polygenic interactions, or some high polymorphic or repetitive regions not optimized by the current method detection. Furthermore, the extraordinarily complex Rho-GAP-GEF signaling network contains many unknown regulators, and it is possible that some of the identified variants affect vascular permeability through pathways that have yet to be identified.
The next steps in the analysis of these data are 2-fold. First, we will continue to locate and enroll new SCLS patients to perform WES to increase the size of our clinical and genetic dataset. Second, we will continue our in vitro investigations of the high priority genes such as DOCK6. Should whole-genome sequencing become more readily available, we may perform this technique on all collected patients to investigate nonprotein coding abnormalities. Finally, we have shown the feasibility of using WES to diagnose critically ill ICU patients. WES is readily available through programs like the Pediatric Genomics Discovery Program at Yale School of Medicine, and should be considered by intensivists and their trainees caring for critically ill patients with clinical symptoms and syndrome suspicious for underlying genetic defects.
SCLS is a rare and potentially lethal clinical phenomenon with a unique yet consistent constellation of signs and symptoms in both pediatric and adult patients. Our study failed to identify a uniform exomic genetic etiology or implicate a single molecular pathway as an underlying cause of SCLS. Other etiologies of SCLS should be considered in future studies, such as nonexomic genetic abnormalities, polygenic interactions, dysregulated inflammatory or vascular permeability signaling, infection, or MGUS. Our WES investigation did identify several candidate genes in individual patients, thereby creating a viable platform for the detection of clinically relevant protein mutations for future in vivo analysis. Finally, through our program's ongoing investigations, we demonstrated that WES may have direct benefits for critically ill patients.
The authors thank the patients and families, who are the inspiration for this study, and also thank the Yale Center for Genome Analysis (YCGA) for exome sequencing support including the assistance by James Knight for sequence alignment algorithms.
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Capillary leak syndrome; critical illness; endothelial junctions; pediatrics; vascular permeability; whole-exome sequencing; CADD; combined annotated dependent depleted; EXaC; exome aggregation consortium; GAP; GTPase-accelerating protein; GEF; guanine nucleotide exchange factors; GnomAD; genome aggregate database; IRB; institutional review board; MAF; minor allele frequency; MGUS; monoclonal gammopathy of undetermined significance; NIAID; National Institute of Allergy and Infectious Disease; NIH; National Institute of Health; PCA; principal component analysis; PGDP; Pediatric Genomics Discovery Program; PPH2; PolyPhen-2; SCLS; systemic capillary leak syndrome; SIFT; Sorting Intolerant From Tolerant; SNV; single nucleotide variant; VEGF; vascular-endothelial growth factor; WES; whole-exome sequencing
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