Patient 1 was referred at the age of 16 months for evaluation of IBD. He was born to a consanguineous Muslim family (parents first degree cousins) and presented at the age of 9 months with fever and diarrhea that were initially attributed to amebiasis. Despite metronidazole treatment his condition deteriorated, and he developed perianal abscesses with multiple fistulas (Figure 1A). Blood tests were significant for anemia (hemoglobin 6.5 gram/dL), hypoalbuminemia (albumin 1.5 gr/dL) and elevated inflammatory markers (CRP 94 mg/L, normal <5 mg/L). Due to the severity of the perianal disease, the patient underwent double barrel protective ileostomy. Despite this intervention, his condition did not improve. At the age of 16 months the patient appearead cachectic. Weight was 7.4 kg (Z score -4), length 73 cm (Z score -2) and head circumference 46 cm (Z score -2). Abdominal exam showed double barrel ileostomy, with severe inflammation in the surrounding skin, and multiple perianal fistulas with deep ulcerations and perianal fissures. Colonoscopy demonstrated patchy areas of severe colonic ulcerations with pseudopolyps, with normal mucosa in between, while an upper endoscopy was unremarkable. Colonic biopsies revealed chronic active inflammation with focal cryptitis (Figure 1B).
Following a course of broad-spectrum antibiotics and nutritional supplementation, the patient was started on metronidazole, mesalamine and azathioprine, and later began adalimumab. Nevertheless, the patient failed to respond to this TNFa antagonist despite adequate drug levels and absence of anti-drug antibodies, thus experiencing primary pharmacodynamic failure, and at the age of 2.5 years developed colo-vesical fistula that required fistulectomy. Follow up colonoscopy showed severe ulcerations at the sigmoid and ascending colon. Anakinra, an IL1 receptor antagonist, was commenced with escalation of the dose up to 3mg/kg daily, though clinical or laboratory responses were not documented. Importantly, the patient did not develop severe or atypical inflammation until the age of 3 years.
Patient 2 was also born to a consanguineous Muslim family (parents first degree cousins) and presented at the age of 1 month with recurrent fevers, non-bloody diarrhea, oral ulcers, poor weight gain, abdominal distention and arthritis. During infancy he developed recurrent perianal abscesses. At the age of 20 months his weight was 9.7 kg (-2 SD), length 81 cm (-1 SD), head circumference 46 cm (-1 SD). Physical exam was noted for abdominal distention and hepatosplenomegaly, along with a draining perianal abscess. Blood tests demonstrated anemia (hemoglobin 8.8 g/dL) and mildly elevated inflammatory markers (ESR 29 mm/hour). Blood cultures were positive for Providencia stuartii and Pseudomonas aeruginosa, which were treated with Piperacillin-Tazobactam for two weeks. Colonoscopy showed patchy colitis, predominantly at the right colon, while an upper endoscopy was unremarkable. Pathologic assessment showed patchy chronic active colitis. The patient temporarily responded to mesalamine, metronidazole and azathioprine, with a 3 kg weight gain over 10 months and normalization of inflammatory markers. However, he continued to suffer from recurrent febrile episodes, oral ulcers, diarrhea, arthritis and perianal abscesses.
Basic immune studies
Immunoglobulin levels, including IgM, IgA and IgG were within normal limits for both patients (Supplemental Table 3). Moreover, immunoglobulin E was also normal for Patient 1. In addition, lymphocyte subset analysis was within normal limits for both patients ,beside slightly elevated CD8+ T cells (Supplemental Table 3). Finally, TREC levels for both patients were within normal limits, reflecting intact thymic function.
Identification of RIPK1 mutation
Following WES analysis, we identified 7,928 and 6,887 homozygous variants for Patients 1 and 2, respectively, that affect protein sequences. These numbers were reduced to 49 and 77 variants, respectively, after filtration for common variants (MAF ≥0.01) in either our local in-house exomes database (n~3500) or external databases such as 1000 Genomes Project (1 KG; https://www.internationalgenome.org/1000-genomes-browsers) or dbSNP 135 database, the NHLBI Exome Sequencing Project (ESP) (http://evs.gs.washington.edu/EVS/) or gnomAD database (https://gnomad.broadinstitute.org/). A c.1934C>T missense mutation residing in Exon 11 was identified in Patient 1, and a c.580G>A RIPK1 missense mutation residing in Exon 4 was identified in Patient 2 (Figure 2). Both mutations were verified using Sanger sequencing, were designated as deleterious to protein function and were not found in gnomAD Database, dbSNP, 1000G and ESP6500, nor in our in-house database.
Abnormal Cytokine Production in RIPK1-deficient Patient
To evaluate changes in the architecture and function of peripheral immune system in one of the RIPK1-deficient patients, we performed mass cytometry (CyTOF) using a panel of 36 antibody markers, and compared the results to in comparison to both maternal and paternal blood samples, non-IBD subjects (controls) and CD patients, An unbiased clustering algorithm (Phenograph) was performed on multiple immune populations. To begin, we clustered on all immune cells (CD45+) and were able to identify 28 unique populations (Figure 3A-C). When comparing abundances of major immune subsets, we identified an increased abundance of T cells, particularly CD8 effector population, in the RIPK1-deficient patient, compared to controls, along with a decrease in all other cell types including monocytes, dendritic cells and B cells (Figure 3 C-E). Interestingly, we also observed a reversal of the CD4 to CD8 ratio compared to all other samples (Figure 3F).
To gain further insight into the functional effects of RIPK1 deficiency, we examined cytokine production by PBMCs following stimulation with either LPS or PMA/Ionomycin, using CyTOF, and clustering on all leukocytes (CD45+ cells, Supplemental Figure 1A-C). We were able to identify 17 unique populations, again demonstrating a CD8 predominance in the patient (Supplemental Figure 1A-B). RIPK1-deficient patient’s immune cells exhibited decreased IL-6 production in response to LPS, but not to PMA-I stimulation, across multiple cell types including T cells, B cells and innate immune cells (Figure 4A). In addition, we observed an increase in IL-22 production across the majority of immune cells, particularly under the LPS stimulation condition (Figure 4B).
Focusing on the individual cell populations, monocytes and dendritic cell’s ability to produce inflammatory cytokines was drastically reduced in the patient, consistent with documented reduced NFkB activity17 (Figure 4C). On the other hand, patient’s T cells produced higher amounts of IFNg than healthy controls upon PMA/I stimulation (Figure 4E). Interestingly, when analyzing immune populations with inhibitory potential, including myeloid derived suppressor cells (MDSC) and CD24+ B cells (containing the putative regulatory B cell populations), we demonstrate higher production of inflammatory cytokines (IL-23, IL1b, and IL-8), but also IL-10, compared with cells obtained from the parents (Figure 4C), suggesting a possible dysfunction of these cells.