Mouse models of neutropenia reveal progenitor-stage-specific defects.

Advances in genetics and sequencing have identified a plethora of disease-associated and disease-causing genetic alterations. To determine causality between genetics and disease, accurate models for molecular dissection are required; however, the rapid expansion of transcriptional populations identified through single-cell analyses presents a major challenge for accurate comparisons between mutant and wild-type cells. Here we generate mouse models of human severe congenital neutropenia (SCN) using patient-derived mutations in the GFI1 transcription factor. To determine the effects of SCN mutations, we generated single-cell references for granulopoietic genomic states with linked epitopes1, aligned mutant cells to their wild-type equivalents and identified differentially expressed genes and epigenetic loci. We find that GFI1-target genes are altered sequentially, as cells go through successive states of differentiation. These insights facilitated the genetic rescue of granulocytic specification but not post-commitment defects in innate immune effector function, and underscore the importance of evaluating the effects of mutations and therapy within each relevant cell state.

Infrequent fractures and resilient bone mineral density: bone health in patients with Fanconi anemia.

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Pre-transplant glomerular hyperfiltration is not a risk factor for increased renal morbidity and mortality in pediatric stem cell transplant patients.

Low glomerular filtration rate (GFR) prior to stem cell transplant (SCT) is associated with increased morbidity and mortality. The implications of abnormally high GFRs, or glomerular hyperfiltration, prior to SCT are unknown. Twenty-two of 74 consecutive pediatric SCT patients over 2 years old at a single center were hyperfiltrating prior to SCT, median nuclear medicine GFR 154 mL/min/1.73 m2 [interquartile range: 146-170]. There was no association between hyperfiltration and any transplant demographics, nor between hyperfiltration and acute kidney injury (p = .8), renal replacement therapy (p = .63), 1-year event-free survival (p = 1), or abnormal creatinine-based estimated GFR at a median follow-up of 4.7 years (p = .73).

Phenotypes of adults with Fanconi anaemia.

The long-term outcomes of adults with Fanconi anaemia (FA) have improved with advances in haematopoietic stem cell transplantation (HSCT) and more detailed follow-up and screening guidelines. The phenotype of those who survive to adulthood may differ from the typical presentation of FA. We collected retrospective clinical data on adults with FA who received their care at the Cincinnati Children's Hospital Medical Center. In our final cohort of 52 patients, there were 29 females and 23 males, with median (range) age of 21 (18-37) years. Overall, 42 patients (81%) were alive at last follow-up. In all, 36 adults (69%) had undergone HSCT, including eight who had developed myelodysplasia or acute myeloid leukaemia. Eight (15%) developed squamous cell carcinoma. Endocrine complications were common, including hypothyroidism (42%), diabetes (10%), low body mass index (31%) and low bone mineral density (51%). The majority of adults with FA were employed (52%) or full-time students (13%). A significant subset of patients with FA are surviving into adulthood without requiring HSCT. Endocrine abnormalities and the development of solid tumours complicate adulthood. With improved survival outcomes following HSCT and more aggressive malignancy screening protocols, ongoing longitudinal analysis will be important to further characterise this cohort and the phenotype of untransplanted adults with FA.

Gonadal function in pediatric Fanconi anemia patients treated with hematopoietic stem cell transplant.

Gonadal dysfunction and reduced fertility are clinical manifestations well described in patients with Fanconi anemia (FA) and following hematopoietic stem cell transplantation (HSCT). It is difficult to differentiate gonadal dysfunction from the primary disease itself or from HSCT procedures. Therefore, it is important to manage expectations about gonadal failure and infertility for all patients with FA, regardless of the HSCT status. We performed a retrospective analysis of 98 pediatric patients with FA who were transplanted between July 1990 and June 2020 to evaluate the incidence of gonadal dysfunction in female and male patients with FA. New-onset premature ovarian insufficiency (POI) was diagnosed in a total of 30 (52.6%) patients. Follicle-stimulating hormone and luteinizing hormone levels were increased in patients diagnosed with POI. Anti- Mullerian hormone levels declined in POI patients after HSCT (r2=0.21; P=0.001). Twenty (48.8%) male patients were diagnosed with testicular failure. Follicle-stimulating hormone levels increased after HSCT even in patients without testicular failure (r2=0.17; P=0.005). Inhibin B levels decreased over time after HSCT in patients with testicular failure (r2=0.14; P=0.001). These data indicate brisk decline in already impaired gonadal function in transplanted children with FA.

Complement blockade for TA-TMA: lessons learned from a large pediatric cohort treated with eculizumab.

Overactivated complement is a high-risk feature in hematopoietic stem cell transplant (HSCT) recipients with transplant-associated thrombotic microangiopathy (TA-TMA), and untreated patients have dismal outcomes. We present our experience with 64 pediatric HSCT recipients who had high-risk TA-TMA (hrTA-TMA) and multiorgan injury treated with the complement blocker eculizumab. We demonstrate significant improvement to 66% in 1-year post-HSCT survival in treated patients from our previously reported untreated cohort with same hrTA-TMA features that had 1-year post-HSCT survival of 16.7%. Responding patients benefited from a brief but intensive course of eculizumab using pharmacokinetic/pharmacodynamic-guided dosing, requiring a median of 11 doses of eculizumab (interquartile range [IQR] 7-20). Treatment was discontinued because TA-TMA resolved at a median of 66 days (IQR 41-110). Subjects with higher complement activation measured by elevated blood sC5b-9 at the start of treatment were less likely to respond (odds ratio, 0.15; P = .0014) and required more doses of eculizumab (r = 0.43; P = .0004). Patients with intestinal bleeding had the fastest eculizumab clearance, required the highest number of eculizumab doses (20 vs 9; P = .0015), and had lower 1-year survival (44% vs 78%; P = .01). Over 70% of survivors had proteinuria on long-term follow-up. The best glomerular filtration rate (GFR) recovery in survivors was a median 20% lower (IQR, 7.3%-40.3%) than their pre-HSCT GFR. In summary, complement blockade with eculizumab is an effective therapeutic strategy for hrTA-TMA, but some patients with severe disease lacked a complete response, prompting us to propose early intervention and search for additional targetable endothelial injury pathways.

Pediatric 129 Xe Gas-Transfer MRI-Feasibility and Applicability.

BACKGROUND: 129 Xe gas-transfer MRI provides regional measures of pulmonary gas exchange in adults and separates xenon in interstitial lung tissue/plasma (barrier) from xenon in red blood cells (RBCs). The technique has yet to be demonstrated in pediatric populations or conditions. PURPOSE/HYPOTHESIS: To perform an exploratory analysis of 129 Xe gas-transfer MRI in children. STUDY TYPE: Prospective. POPULATION: Seventy-seven human volunteers (38 males, age = 17.7 ± 15.1 years, range 5-68 years, 16 healthy). Four pediatric disease cohorts. FIELD STRENGTH/SEQUENCE: 3-T, three-dimensional-radial one-point Dixon Fast Field Echo (FFE) Ultrashort Echo Time (UTE). ASSESSMENT: Breath hold compliance was assessed by quantitative signal-to-noise and dynamic metrics. Whole-lung means and standard deviations were extracted from gas-transfer maps. Gas-transfer metrics were investigated with respect to age and lung disease. Clinical pulmonary function tests were retrospectively acquired for reference lung disease severity. STATISTICAL TESTS: Wilcoxon rank-sum tests to compare age and disease cohorts, Wilcoxon signed-rank tests to compare pre- and post-breath hold vitals, Pearson correlations between age and gas-transfer metrics, and limits of normal with a binomial exact test to compare fraction of subjects with abnormal gas-transfer. P ≤ 0.05 was considered significant. RESULTS: Eighty percentage of pediatric subjects successfully completed 129 Xe gas-transfer MRI. Gas-transfer parameters differed between healthy children and adults, including ventilation (0.75 and 0.67) and RBC:barrier ratio (0.31 and 0.46) which also correlated with age (ρ = -0.76, 0.57, respectively). Bone marrow transplant subjects had impaired ventilation (90% of reference) and increased dissolved 129 Xe standard deviation (242%). Bronchopulmonary dysplasia subjects had decreased barrier-uptake (69%). Cystic fibrosis subjects had impaired ventilation (91%) and increased RBC-transfer (146%). Lastly, childhood interstitial lung disease subjects had increased ventilation heterogeneity (113%). Limits of normal provided detection of abnormalities in additional gas-transfer parameters. DATA CONCLUSION: Pediatric 129 Xe gas-transfer MRI was adequately successful and gas-transfer metrics correlated with age. Exploratory analysis revealed abnormalities in a variety of pediatric obstructive and restrictive lung diseases. LEVEL OF EVIDENCE: 2 TECHNICAL EFFICACY STAGE: 2.

Coronavirus disease 2019 and vaccination in patients with Shwachman-Diamond syndrome.

Because they can experience neutropenia due to bone marrow failure, patients with Shwachman-Diamond syndrome (SDS) carry increased risk for serious infections compared with the general population; however, there has been a paucity of data on the incidence and severity of coronavirus disease 2019 (COVID-19) in patients with SDS. We compiled results from a survey distributed to participants in the SDS Registry in May-June 2021. In this report, we describe the characteristics and outcomes of patients with SDS who had COVID-19. Patients reported a short clinical course without significant complications or cytopenias. Additionally, COVID-19 vaccines were well tolerated with minor side effects.

Pain, depressive symptoms, and health-related quality of life among survivors of pediatric hematopoietic stem cell transplant.

OBJECTIVE: Pediatric hematopoietic stem cell transplant (HCT) is an intensive medical procedure associated with significant late effects, of which pain is a prominent example. While pain is associated with increased depressive symptoms and health-related quality-of-life (HRQoL) impairments in other pediatric chronic illness populations, associations between these variables are not well understood in pediatric HCT. Clarifying these associations may inform clinical interventions to improve health outcomes following pediatric HCT. This study aimed to investigate the relations between pain intensity, depressive symptoms, and HRQoL in survivors of pediatric HCT. METHOD: Fifty-one survivors of pediatric HCT (Mage  = 14.3 years, standard deviation [SD] = 4.3; 58.8% male; 80.4% White) completed self-report measures of pain intensity, depressive symptoms, and HRQoL. Demographic and disease information was collected via demographic forms and medical record review. Path analysis was used to examine hypothesized associations between pain intensity, depressive symptoms, and HRQoL. RESULTS: Analyses revealed direct effects of pain intensity on depressive symptoms (estimate [Est.] = .23, p < .001) and HRQoL (Est. = -.2, p = .04), and direct effects of depressive symptoms on HRQoL (Est. = -.68, p < .001). Depressive symptoms also mediated the relationship between pain intensity and HRQoL (Est. = -.16, p = .006). CONCLUSIONS: Greater pain intensity was associated directly with increased depressive symptoms and indirectly with HRQoL through depressive symptoms. Results of this study suggest that multitargeted cognitive behavioral interventions that address pain and depressive symptoms may improve HRQoL ratings in survivors of pediatric HCT.

Neutropenia and intellectual disability are hallmarks of biallelic and de novo CLPB deficiency.

PURPOSE: To investigate monoallelic CLPB variants. Pathogenic variants in many genes cause congenital neutropenia. While most patients exhibit isolated hematological involvement, biallelic CLPB variants underlie a neurological phenotype ranging from nonprogressive intellectual disability to prenatal encephalopathy with progressive brain atrophy, movement disorder, cataracts, 3-methylglutaconic aciduria, and neutropenia. CLPB was recently shown to be a mitochondrial refoldase; however, the exact function remains elusive. METHODS: We investigated six unrelated probands from four countries in three continents, with neutropenia and a phenotype dominated by epilepsy, developmental issues, and 3-methylglutaconic aciduria with next-generation sequencing. RESULTS: In each individual, we identified one of four different de novo monoallelic missense variants in CLPB. We show that these variants disturb refoldase and to a lesser extent ATPase activity of CLPB in a dominant-negative manner. Complexome profiling in fibroblasts showed CLPB at very high molecular mass comigrating with the prohibitins. In control fibroblasts, HAX1 migrated predominantly as monomer while in patient samples multiple HAX1 peaks were observed at higher molecular masses comigrating with CLPB thus suggesting a longer-lasting interaction between CLPB and HAX1. CONCLUSION: Both biallelic as well as specific monoallelic CLPB variants result in a phenotypic spectrum centered around neurodevelopmental delay, seizures, and neutropenia presumably mediated via HAX1.

Gastrointestinal Hemorrhage: A Manifestation of the Telomere Biology Disorders.

OBJECTIVE: To describe the clinical features, therapeutic interventions, and patient outcomes of gastrointestinal (GI) hemorrhage in individuals with a telomere biology disorder, including dyskeratosis congenita, Hoyeraal-Hreidarsson syndrome, Revesz syndrome, and Coats plus. STUDY DESIGN: Clinical Care Consortium for Telomere Associated Ailments members were invited to contribute data on individuals with telomere biology disorders at their institutions who experienced GI bleeding. Patient demographic, laboratory, imaging, procedural, and treatment information and outcomes were extracted from the medical record. RESULTS: Sixteen patients who experienced GI hemorrhage were identified at 11 centers. Among 14 patients who underwent genetic testing, 8 had mutations in TINF2, 4 had mutations in CTC1 or STN1, and 1 patient each had a mutation in TERC and RTEL1. Ten patients had a history of hematopoietic cell transplantation. The patients with Coats plus and those without Coats plus had similar clinical features and courses. Angiodysplasia of the stomach and/or small bowel was described in 8 of the 12 patients who underwent endoscopy; only 4 had esophageal varices. Various medical interventions were trialed. No single intervention was uniformly associated with cessation of bleeding, although 1 patient had a sustained response to treatment with bevacizumab. Recurrence was common, and the overall long-term outcome for affected patients was poor. CONCLUSIONS: GI bleeding in patients with telomere biology disorders is associated with significant morbidity and with vascular ectasias rather than varices.

Testicular thrombotic microangiopathy: An unrecognized complication.

Thrombotic microangiopathy (TMA) causes global endothelial damage and multiorgan injury. No study has described reproductive organ involvement in TMA. Our study aimed to characterize testicular involvement in TMA. We reviewed autopsies from four patients who underwent hematopoietic cell transplant (HCT) complicated by TMA. Three patients had striking histologic evidence of TMA, while the fourth had normal testicular histology. This suggests that TMA injures the testicles and may adversely affect fertility. There is now an urgent need for a larger analysis of reproductive organ involvement in TMA.

α4ß7 Integrin expression and blockade in pediatric and young adult gastrointestinal graft-versus-host disease.

BACKGROUND: We hypothesized that α4ß7 integrin expression on effector memory T cells (TEMs) would be elevated in pediatric hematopoietic stem cell transplant (HSCT) patients before and at diagnosis of acute gastrointestinal graft-versus-host disease (GI GVHD) symptoms compared to patients without GVHD, and that clinical blockade of α4ß7 integrin with vedolizumab would be effective in pediatric GI GVHD. METHODS: We analyzed surface expression of α4ß7 integrin on T cells from 48 pediatric allogeneic HSCT recipients from our biorepository with known clinical outcomes as follows: acute GI GVHD (n = 22), isolated skin GVHD (n = 12), and no GVHD (n = 14). T-cell analyses were performed 1 week before and at GVHD diagnosis in patients with GVHD, and day +30 after HSCT in patients without GVHD. We describe clinical outcomes of seven additional patients, different from above-described 48 patients, who received vedolizumab (anti-α4ß7 integrin antibody) for the treatment of steroid-refractory acute GI GVHD. RESULTS: Expression of α4ß7 integrin on CD8+ TEMs was upregulated in patients with GI GVHD compared to the no GI GVHD (skin GVHD + no GVHD) group 1 week prior to clinical symptoms (p = .02) and at acute GI GVHD diagnosis (p = .05). Four of seven treated patients with clinical steroid-refractory acute GI GVHD were evaluable for response to vedolizumab. One patient had a complete response at day +28, while two had a partial response, and one had no response. No adverse effects directly attributable to vedolizumab were observed. CONCLUSION: Our data suggest a rationale for the blockade of α4ß7 integrin for acute GI GVHD management in children.

Incidence of thyroid dysfunction in children after HSCT with reduced intensity conditioning (RIC) or myeloablative conditioning (MAC).

We have previously demonstrated a 11% incidence of post-transplant de novo thyroid disease, even with a radiation-free RIC regimen. Following the enactment of a universal late effects screening program at our institution, we compared the outcomes of 108 pediatric hematopoietic stem cell transplant recipients after a RIC regimen (n = 33) to those after a MAC regimen (n = 75) during the same time period. Overall, 10% of subjects developed thyroid dysfunction after HSCT, with a median follow-up of 669 days. Seven subjects had primary hypothyroidism prior to HSCT. Of the thirty-one subjects who received RIC, one (3.2%) developed a new thyroid disorder, compared to the nine of sixty-nine (13.0%) subjects who received MAC (p = .167). No significant associations were seen with donor type, graft-vs.-host disease, or total body irradiation. Nine of the 10 subjects who developed thyroid disease after transplant were asymptomatic. Continued follow-up of this contemporary cohort will further delineate risk factors for post-transplant-associated thyroid dysfunction and better inform discussions of transplant-associated sequelae.

Thinking Beyond HLH: Clinical Features of Patients with Concurrent Presentation of Hemophagocytic Lymphohistiocytosis and Thrombotic Microangiopathy.

Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of excessive immune system activation driven mainly by high levels of interferon gamma. The clinical presentation of HLH can have considerable overlap with other inflammatory conditions. We present a cohort of patients with therapy refractory HLH referred to our center who were found to have a simultaneous presentation of complement-mediated thrombotic microangiopathy (TMA). Twenty-three patients had therapy refractory HLH (13 primary, 4 EVB-HLH, 6 HLH without known trigger). Sixteen (69.6%) met high-risk TMA criteria. Renal failure requiring renal replacement therapy, severe hypertension, serositis, and gastrointestinal bleeding were documented only in patients with HLH who had concomitant complement-mediated TMA. Patients with HLH and without TMA required ventilator support mainly due to CNS symptoms, while those with HLH and TMA had respiratory failure predominantly associated with pulmonary hypertension, a known presentation of pulmonary TMA. Ten patients received eculizumab for complement-mediated TMA management while being treated for HLH. All patients who received the complement blocker eculizumab in addition to the interferon gamma blocker emapalumab had complete resolution of their TMA and survived. Our observations suggest co-activation of both interferon and complement pathways as a potential culprit in the evolution of thrombotic microangiopathy in patients with inflammatory disorders like refractory HLH and may offer novel therapeutic approaches for these critically ill patients. TMA should be considered in children with HLH and multi-organ failure, as an early institution of a brief course of complement blocking therapy in addition to HLH-targeted therapy may improve clinical outcomes in these patients.

Somatic mutations and clonal hematopoiesis in congenital neutropenia.

Severe congenital neutropenia (SCN) and Shwachman-Diamond syndrome (SDS) are congenital neutropenia syndromes with a high rate of leukemic transformation. Hematopoietic stressors may contribute to leukemic transformation by increasing the mutation rate in hematopoietic stem/progenitor cells (HSPCs) and/or by promoting clonal hematopoiesis. We sequenced the exome of individual hematopoietic colonies derived from 13 patients with congenital neutropenia to measure total mutation burden and performed error-corrected sequencing on a panel of 46 genes on 80 patients with congenital neutropenia to assess for clonal hematopoiesis. An average of 3.6 ± 1.2 somatic mutations per exome was identified in HSPCs from patients with SCN compared with 3.9 ± 0.4 for healthy controls (P = NS). Clonal hematopoiesis due to mutations in TP53 was present in 48% (13/27) of patients with SDS but was not seen in healthy controls (0/17, P < .001) or patients with SCN (0/40, P < .001). Our SDS cohort was young (median age 6.3 years), and many of the patients had multiple TP53 mutations. Conversely, clonal hematopoiesis due to mutations of CSF3R was present in patients with SCN but was not detected in healthy controls or patients with SDS. These data show that hematopoietic stress, including granulocyte colony-stimulating factor, do not increase the mutation burden in HSPCs in congenital neutropenia. Rather, distinct hematopoietic stressors result in the selective expansion of HSPCs carrying specific gene mutations. In particular, in SDS there is enormous selective pressure to expand TP53-mutated HSPCs, suggesting that acquisition of TP53 mutations is an early, likely initiating event, in the transformation to myelodysplastic syndrome/acute myeloid leukemia in patients with SDS.

Inflammatory manifestations in patients with Shwachman-Diamond syndrome: A novel phenotype.

Shwachman-Diamond syndrome (SDS) is an autosomal recessive multisystem disorder characterized by exocrine pancreatic dysfunction, bone marrow failure, and leukemia predisposition. Approximately 90% of cases are due to biallelic mutations in the Shwachman-Bodian-Diamond (SBDS) gene. Additional phenotypic features variably associated with SDS include skeletal, neurologic, hepatic, cardiac, endocrine, and dental abnormalities. We report five subjects with SDS who developed a range of inflammatory manifestations. Three patients developed inflammatory eye conditions. Single cases of juvenile idiopathic arthritis, chronic recurrent multifocal osteomyelitis, and scleroderma were also noted. Clinical presentation and treatment responses are described. Proteomic analysis revealed increased inflammatory signatures in SDS subjects as compared to controls. Treatment of inflammatory manifestations in patients with SDS may be complicated by potential myelosuppressive toxicities of anti-rheumatic medications. Further research is needed to better understand the potential link between inflammatory disorders and SDS to inform effective treatment strategies.

Pediatric and Young Adult Vulvovaginal Graft-versus-Host Disease.

Vulvovaginal graft-versus-host disease (GVHD) is an underdiagnosed and poorly recognized complication of hematopoietic stem cell transplantation (HSCT). Previous studies have reported findings restricted to predominantly adult populations. We report a case series of pediatric and young adult vulvovaginal GVHD, which was identified in 19 patients (median age, 11.8 years; range, 2.4 to 21.9 years) out of a total 302 female patients who underwent transplantation over an 8-year period at a pediatric HSCT center. The majority of patients had concomitant nongenital GVHD; only 1 patient had isolated vulvovaginal GVHD. The median time from bone marrow transplantation to diagnosis of vulvovaginal GVHD was 30 months (range, 2.3 to 97.5 months). A high percentage of the patients in our series were without vulvar or vaginal symptoms (n = 8; 42%), even though 17 patients (89%) presented with grade 3 disease based on current adult grading scales. Vulvar examination findings most frequently included interlabial and clitoral hood adhesions (89%), loss of architecture of the labia minora or clitoral hood (42%), and skin erosions or fissures (37%). Only 5 patients underwent a speculum exam, none of whom had vaginal GVHD. Examination findings of primary ovarian insufficiency (POI) can overlap with those of GVHD, and 6 patients (32%) in our cohort were diagnosed with POI. Only 1 patient was on systemic hormone replacement therapy at the time of vulvovaginal GVHD diagnosis. The majority of patients (n = 16) were treated with topical steroid therapy, with a median time to response of 43 days. Five patients (26%) had a complete response to therapy, and 10 patients (53%) had a partial response. This case series provides valuable insight into pediatric and young adult vulvovaginal GVHD and highlights the need for increased screening for vulvar disease in this population.

Understanding the evolving phenotype of vascular complications in telomere biology disorders.

Vascular complications such as bleeding due to gastrointestinal telangiectatic anomalies, pulmonary arteriovenous malformations, hepatopulmonary syndrome, and retinal vessel abnormalities are being reported in patients with telomere biology disorders (TBDs) more frequently than previously described. The international clinical care consortium of telomere-associated ailments and family support group Dyskeratosis Congenita Outreach, Inc. held a workshop on vascular abnormalities in the TBDs at the National Cancer Institute in October 2017. Clinicians and basic scientists reviewed current data on vascular complications, hypotheses for the underlying biology and developed new collaborations to address the etiology and clinical management of vascular complications in TBDs.