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BACKGROUND: Prospective genetic evaluation of patients at our referral research hospital presents clinical research challenges. OBJECTIVE: This study sought not only a single-gene explanation for participants’ immune-related presentations, but viewed each participant holistically, with the potential to have multiple genetic contributions to their immune-phenotype and other heritable comorbidities relevant to their presentation and health. METHODS: We developed a program integrating exome sequencing, chromosomal microarray, phenotyping, results return with genetic counseling, and reanalysis in 1505 individuals from 1000 families with suspected or known inborn errors of immunity. RESULTS: Probands were 50.8% female, 71.5% ≥18 years, and had diverse immune presentations. Overall, 327/1000 probands (32.7%) received 361 molecular diagnoses. These included 17 probands with diagnostic copy number variants, 32 probands with secondary findings, and 31 probands with multiple molecular diagnoses. Reanalysis added 22 molecular diagnoses, predominantly due to new disease-gene associations (9/22, 40.9%). One-quarter of the molecular diagnoses (92/361) did not involve immune-associated genes. Molecular diagnosis was correlated with younger age, male sex, and a higher number of organ systems involved. This program also facilitated the discovery of new gene-disease associations such as SASH3-related immunodeficiency. A review of treatment options and ClinGen actionability curations suggest that at least 251/361 (69.5%) of these molecular diagnoses could translate into ≥1 management option. CONCLUSION: This program contributes to our understanding of the diagnostic and clinical utility whole exome analysis on a large scale.

COVID-19 vaccine research in immune-deficient/disordered people (IDP) has primarily focused on cancer and organ transplantation populations. We followed 195 IDP with varied immune disorders and 35 healthy volunteers (HV) from April 2021-April 2022. Anti-spike IgG and angiotensin-converting enzyme 2 pseudo-neutralization were measured though six months post-dose 3. Anti-spike IgG was detected in 93% of IDP by six months post-dose 3. IgG dynamics in IDP and HV were similar, though median IgG levels for IDP were <33% of HV at all timepoints. IgG concentrations were lower against Omicron BA.1 than other variants at all timepoints. Pseudo-neutralization capacity was modestly correlated with anti-spike IgG concentration, but was especially low for Omicron BA.1. Post-vaccination adverse events were minimal. Results were largely unaffected by participants’ immunomodulatory medication and treatments. COVID-19 vaccines are safe, induce anti-spike IgG in most IDP, and should be more strongly recommended for people with immunodeficiencies.

X-linked chronic granulomatous disease (X-CGD) is a rare monogenetic immunodeficiency primarily affecting phagocytes. Precipitated by mutations in the CYBB gene, patients exhibit a compromised oxidative burst, leading to recurrent infections which can be life-threatening. Curiously, autoimmune manifestations are also common in patients and carriers. Here, exploiting the cell type-specific nature of this disorder, we characterize X-CGD on a transcriptional level using single-cell sequencing. Peripheral blood from 14 X-CGD probands and 10 carriers signed onto IRB approved protocol NCT00404560, as well as from 15 controls was sampled, and PBMCs and isolated monocytes were subjected to single-cell sequencing. Probands exhibited a strong differential expression signal relative to controls. This was composed of not only genes previously described to be up-regulated in X-CGD such as IFI27, and indeed an autoimmunity-associated broader type I interferon response, but also previously undescribed genes involved in monocyte function (ARG1), antimicrobial proteins (CAMP, SLPI), and inflammasome components (AIM2). Surprisingly, expression variability was not greater in carriers relative to probands or controls, indicating a lack of cell autonomous effects from the deletion of CYBB. Interestingly, aggregate expression of differentially expressed genes in the probands was able to classify carriers from sex-matched controls with high accuracy (AUROC = 0.92), indicating the presence of an X-CGD-specific gene signature. This gene signature was also strongly co-expressed across 17 chordate species, pointing towards the disruption of ancestral pathways important in antimicrobial immunity in X-CGD probands and carriers. This work was partially supported by a Swiss National Science Foundation fellowship to S.M.

Introduction Chronic granulomatous disease (CGD) is a rare immunodeficiency caused by mutations in the NADPH oxidase complex. Dysregulated immune function may cause inflammatory bowel disease (IBD). Patients with CGD-associated IBD may not respond to or may develop serious infections as a result of traditional IBD therapies such as vedolizumab and infliximab. Ustekinumab is approved for use in Crohn’s disease and ulcerative colitis however there is scarce data on its efficacy and safety in CGD. Aims To evaluate the efficacy and safety of ustekinumab for CGD-associated IBD. Methods A retrospective chart review was conducted on CGD patients followed at a single center who had consented to participate in a natural history study. Clinical, laboratory, and endoscopic data were extracted in those that had received ustekinumab for IBD. Results Eight patients were found. Four were male and four were female. Five were white, one was Asian, one was black, and one was mixed race. Median age at diagnosis of CGD was 3 years (IQR 8) and of IBD was 15.5 years (IQR 20). Median age at initiation of ustekinumab was 27.5 years (IQR 14) and median duration on ustekinumab was 10 months (IQR 7). Six had colonic disease, two had ileocolonic disease, and six had perianal disease. Six failed other biologics (n=5 for vedolizumab, n=1 for infliximab, n=1 for adalimumab). Six patients symptomatically improved whereas two had no improvement. Changes in hemoglobin and C-reactive protein were equivocal. Three patients had improved endoscopic findings, two had unimproved findings, and three patients lacked this data. Overall, four patients achieved clinical remission. However, none of the five patients with endoscopic reevaluation achieved endoscopic remission. Three patients discontinued therapy due to lack of response: two required surgery and one underwent stem cell transplant. Fungal pneumonia (n=2), otitis media (n=1), oral herpes simplex virus 1 (n=1), and viral gastroenteritis (n=1) were reported. One infusion reaction occurred. Discussion In our cohort of eight patients with CGD-associated IBD receiving ustekinumab, results were mixed with four patients experiencing some degree of clinical or endoscopic improvement including four who achieved clinical remission. Multiple CGD-related variables may account for the mixed laboratory findings. Four of the five patients with endoscopic reevaluation had pre-existing strictures that would be unlikely to reverse with medical therapy alone. Of these, two had otherwise resolved endoscopic inflammation. Only two patients had no endoscopic improvement. Two serious infections occurred however CGD confers increased infectious susceptibility and no infections lead to discontinuation of therapy. Given these promising results, further formalized study of ustekinumab in CGD-associated IBD is needed.

Chronic granulomatous disease (CGD) is a rare disorder caused by genetic mutations of the nicotinamide adenine dinucleotide phosphate oxidase complex (NADPH), occurring in approximately 1/200,000 individuals. These mutations decrease residual reactive oxygen species (ROS) levels, leading to dysregulated inflammation. Inflammatory manifestations can be widespread, including severe and recurrent infections. The gastrointestinal tract is the most commonly affected organ with resultant inflammatory bowel disease, termed CGD colitis. Manifestations include abdominal pain, diarrhea with or without blood, nausea/vomiting, obstructions, and fistulas which can occur in a perianal distribution. Patients are often misdiagnosed with Crohn’s disease or ulcerative colitis, especially in the absence of extensive infectious history. We aimed to characterize the small bowel involvement in CGD. Data is presented from a combined retrospective and ongoing prospective observational study of patients with genetically-confirmed CGD who underwent wireless video capsule endoscopy (VCE) at the National Institutes of Health Clinical Center (n = 8). VCEs were performed for clinical indications including abdominal pain (88%), diarrhea (75%), bloody stools (38%), and/or nausea/vomiting (25%). One patient (13%) underwent VCE for otherwise unexplained high inflammatory markers. Laboratory evaluation was significant for leukopenia/leukocytosis (75%), anemia (63%), and elevated C reactive-protein levels (63%). Seven patients (88%) had prior small bowel imaging, however none showed evidence of any abnormality in this organ. The most common VCE findings were ulcers and/or erosions (88%). Most patients also displayed other mucosal changes consistent with inflammation such as erythema and/or edema (88%). There was also evidence of blood or hematin on 63% of the endoscopies. While therapies for CGD colitis are targeted towards colonic involvement, our findings show that the vast majority of symptomatic patients also have active small bowel disease including ulcers, erosions, evidence of bleeding, and other signs of inflammation. These findings, however, are not specific to CGD. Given that certain biologic medications used for Crohn’s disease and ulcerative colitis have been shown to increase the risk of life-threatening infections in patients with CGD, it is important to keep other forms of IBD, especially CGD-related IBD, in mind when interpreting small bowel capsule endoscopy in patients with suspected IBD. Lastly, in patients with confirmed CGD colitis, small bowel disease should be rigorously investigated, and therapy should also seek to address small bowel involvement. Of note, our patients did not display any radiographic abnormalities of the small bowel. Due to our small sample size, we aim to study additional patients in the future to augment our data.

Background: Mutations in the zinc finger transcription factor GATA2 are responsible for: MonoMAC, monocytopenia with nontuberculous mycobacterial (NTM) infections; DCML, dendritic cell, monocyte and lymphoid cell deficiency; Emberger's syndrome with lymphedema and monosomy 7; and familial myelodysplastic syndrome (MDS)/acute myelogenous leukemia (AML). Allogeneic hematopoietic stem cell transplant (HSCT) represents the only definitive therapy for GATA2 deficiency. Methods: Eleven patients with GATA2 deficiency received a myeloablative-conditioning regimen (2 matched related donors or MRD, 4 matched unrelated donors or URD, and 5 haploidentical related donors. MRD and URD received busulfan 3.2 mg/kg/day and fludarabine 40 mg/m2/day on days -6, -5, -4, and -3. Haploidentical related donors received cyclophosphamide 14.5 mg/kg on day's -6 and -5, fludarabine 30 mg/m2/day on day's -6 to -2, busulfan 3.2 mg/kg/day on day's -4 and -3, and 200 cGy TBI on day -1. MRD and URD recipients received tacrolimus and short course methotrexate post-transplant, while haploidentical related donor recipients received cyclophosphamide 50 mg/kg/day on days + 3 and +4 followed by tacrolimus and mycophenolate mofetil as post-transplant immunosuppression for graft-versus-host disease. Results: Ten of the 11 (91%) of patients are alive and disease-free at a mean follow-up of 12 months (range 1 mo to 24 mo). One URD recipient died from persistent acute myelogenous leukemia. Four patients developed graft-versus-host disease, one case Grade 4. All 10 patients who survived had complete reconstitution of the monocyte, NK, and B-lymphocyte compartments, the three cell compartments that were severely deficient pre-transplant. All 10 patients had reversal of the infection susceptibility phenotype. In particular, there were no recurrences of NTM infections. Importantly, all 10 patients had correction of the cytogenetic abnormalities present pre-transplant (5 patients with trisomy 8 and 1 patient with monosomy 7). Conclusions: Myeloablative HSCT in GATA2 deficiency results in uniform engraftment and reversal of the hematologic, cytogenetic, and clinical manifestations of GATA2 deficiency. There was a low regimen-related toxicity, even in this cohort of patients with considerable co-morbidities. We anticipate that with HSCT earlier in the clinical course, before significant organ damage or clonal evolution of MDS to AML or CMML occurs, the outcome of allogeneic HSCT in patients with GATA2 deficiency will continue to improve. Haploidentical related donor transplant appears to be particularly well suited for this disease, especially when the disease presents as a hypocellular myelodysplastic syndrome. Disclosures No relevant conflicts of interest to declare.

Background Heterozygous sporadic or inherited mutations in the GATA2 gene result in a syndrome known variably as: “MonoMAC” for monocytopenia with nontuberculous mycobacterial (NTM) infections; DCML, dendritic cell, monocyte and lymphoid cell deficiency; Emberger's syndrome, lymphedema and MDS with monosomy 7; and familial myelodysplastic syndrome (MDS)/acute myelogenous leukemia (AML). Life-threatening opportunistic infections and myeloid transformation constitute the rationale for allogeneic hematopoietic stem cell transplant (HSCT) for GATA2 deficiency. Methods We treated 14 patients with GATA2 deficiency using a nonmyeloablative-conditioning regimen and matched related donors (MRD) (n=4), matched unrelated donors (URD) (n=4), umbilical cord blood (UCB) (n=4), and haploidentical related donor (HD) (n=2) sources. There was considerable pre-transplant morbidity in this cohort of patients. MRD and URD recipients received 200 cGy of total body irradiation (TBI) and 3 days of fludarabine; UCB recipients received 200cGy TBI, cyclophosphamide 50 mg/kg on day -6, and 5 days of fludarabine; HD recipients received 200cGy TBI, cyclophosphamide 14.5 mg/kg on days -6 and -5, and fludarabine for 5 days. MRD, URD, and UCB recipients received tacrolimus and sirolimus for GVHD prophylaxis. HD recipients received cyclophosphamide 50 mg/kg on days + 3 and +4 followed by tacrolimus and mycophenolate mofetil. MRD and URD donor recipients received peripheral blood stem cells (PBSC) and HD donor recipients received bone marrow stem cells. Results (table 1): The median follow-up in the MRD cohort was 32 months, excluding one early death in a patient on a ventilator at the time of transplant. One patient relapsed one year post-transplant and required re-transplant using a myeloablative regimen. All 4 patients in this cohort developed acute GVHD. In the URD cohort, all patients are alive, however one patient rejected the PBSC graft and required a second URD transplant from a different donor. Two patients developed acute GVHD. In the UCB cohort, there was one early death from sepsis, one graft rejection, and one donor cell leukemia that occurred 2.5 years post-transplant. The remaining patient had a complicated course, but at 3 years post-transplant he is fully engrafted, on no medications, and has no GVHD. In the two patients who received HD transplants, one had progressed to proliferative CMML prior to transplant and died in the immediate post-transplant period. The second patient engrafted and is doing well two months post-transplant with resolution of an EBV-driven T cell lymphoma. All patients who engrafted had complete reconstitution of the monocyte, NK, and B lymphocyte compartments; all had correction of the underlying myeloid malignancy, and reversal of the infection susceptibility phenotype, characteristic of the disease. In particular, there were no recurrences of NTM. Conclusions Nonmyeloablative HSCT in GATA2 deficiency results in reconstitution of the severely deficient monocyte, B and NK cell populations and correction of the infection susceptibility phenotype. However, 1 of 4 MRD recipients developed a relapse of the original clone, and 1 of 4 URD recipients rejected the donor PBSC. The incidence of relapse and rejection, as well as the unfavorable cytogenetics in many patients, suggests that a more intense conditioning regimen is required to treat these patients. In this regard, we are now using a myeloablative regimen with busulfan and fludarabine. The poor outcome with UCB in this cohort of immunocompromised patients supports the use of HD transplants when a MRD or URD is not available. We anticipate that with increasing use of genetic testing for GATA2 mutations, patients will be transplanted earlier in the course of disease, before significant organ damage or clonal evolution of MDS to AML and CMML occurs, and that the outcome of allogeneic HSCT in these patients will continue to improve with these modifications in the transplant approach. Disclosures: No relevant conflicts of interest to declare.

Abstract 3091 Background: Recently, sporadic and heritable mutations in the zinc finger transcription factor GATA2 were shown to be responsible for four different syndromes in young adults coupling opportunistic infection with a predilection to develop myelodysplastic syndrome (MDS)/acute myelogenous leukemia (AML). These four syndromes are: MonoMAC, monocytopenia with nontuberculous mycobacterial (NTM) infections; DCML, dendritic cell, monocyte and lymphoid cell deficiency; Emberger's syndrome, lymphedema and MDS with monosomy 7; and familial MDS/AML. Life-threatening infections, and the transformation to AML, either alone or together, constitute a rationale for allogeneic hematopoietic stem cell transplant (HSCT) for GATA2 deficiency. Methods: We evaluated matched related, unrelated, and umbilical cord blood as donor sources for nonmyeloablative conditioning for HSCT in GATA2 deficiency. Twelve patients with GATA2 deficiency underwent allogeneic transplant: 4 received peripheral blood stem cells (PBSCs) from matched related-donors (MRD), 4 received PBSC from matched unrelated-donors (MUD), and 4 received umbilical cord blood (UCB) units. Recipients of MRD and MUD transplant received fludarabine and 200cGy of total body irradiation (TBI), while UCB recipients received cyclophosphamide 50 mg/kg, fludarabine, and 200cGy of TBI. All patients received tacrolimus and sirolimus for GVHD prophylaxis. This cohort of patients had considerable pre-transplant morbidity: two patients required baseline oxygen for pulmonary alveolar proteinosis, one of whom was on a ventilator at the time of transplant; one patient had active hepatitis C that was not responding to therapy; one patient had RAEB-2; two patients were platelet transfusion-dependent; one patient had recurrent strokes and culture negative endocarditis two months before transplant. Results: Median follow-up for patients was 14.4 months (range 0.2–38.2 months). Ten of 11 patients engrafted at a median of 10 days (range 0–76); engraftment was not evaluable in a recipient of an UCB transplant due to death early in the post-transplant period. One rejection occurred in a recipient of a double UCB transplant who had been heavily transfused pre-transplant. All patients who engrafted had complete reconstitution of the monocyte, NK, and B-lymphocyte compartments, the three cell compartments that were severely deficient pre-transplant, and all had reversal of the infection susceptibility phenotype, characteristic of the disease. In particular, there were no recurrences of NTM infection and extensive human papilloma virus infections regressed starting around 6 months post transplant. Two patients required a single cycle of pre-transplant chemotherapy for RAEB-1 and RAEB-2, respectively. In both patients the monosomy 6 and monosomy 7 clones have not recurred, now 2.5 years and 9 months following single UCB and MUD transplant, respectively. Three patients died, two early after transplant. One recipient of UCB, who had pre-transplant hepatitis C, died on day+7 of fulminant liver failure and sepsis. A second patient, who was intubated at the time of transplant because of severe pulmonary alveolar proteinosis and pulmonary hypertension, died on day + 88 of grade IV GVHD after MRD transplant. The third patient died 9 months after transplant of sepsis. One recipient of a MRD relapsed from her MDS at 1 year following transplant and was retransplanted using a myeloablative conditioning regimen. She is alive with no evidence of MDS at 3 months. Acute GVHD developed in 6 patients, five of which were steroid-responsive. One patient developed chronic GVHD. Other complications included immune mediated cytopenias that responded to rituximab and eltrombopag. Conclusions: Nonmyeloablative HSCT in GATA2 deficiency results in reconstitution of the severely deficient monocyte, B and NK cell populations and reversal of the infection susceptibility phenotype. The TRM in this cohort of patients with severe comorbidities was 25%. Now that genetic testing for GATA2 mutations is available, we anticipate that earlier diagnosis will enable patients to be transplanted earlier in the course of disease, before significant organ damage or clonal evolution of MDS to AML. Disclosures: No relevant conflicts of interest to declare.