O’Byrne, Megan M.

Congenital heart diseases (CHDs), including hypoplastic left heart syndrome (HLHS), are genetically complex and poorly understood. Here, a multidisciplinary platform was established to functionally evaluate novel CHD gene candidates, based on whole-genome and iPSC RNA sequencing of a HLHS family-trio. Filtering for rare variants and altered expression in proband iPSCs prioritized 10 candidates. siRNA/RNAi-mediated knockdown in healthy human iPSC-derived cardiomyocytes (hiPSC-CM) and in developing Drosophila and zebrafish hearts revealed that LDL receptor-related protein LRP2 is required for cardiomyocyte proliferation and differentiation. Consistent with hypoplastic heart defects, compared to parents the proband’s iPSC-CMs exhibited reduced proliferation. Interestingly, rare, predicted-damaging LRP2 variants were enriched in a HLHS cohort; however, understanding their contribution to HLHS requires further investigation. Collectively, we have established a multi-species high-throughput platform to rapidly evaluate candidate genes and their interactions during heart development, which are crucial first steps toward deciphering oligogenic underpinnings of CHDs, including hypoplastic left hearts.

Background: Annual rankings by US News and World Report are a widely utilized metric by both health care leaders and patients. One longstanding measure is time to treatment of femur shaft fractures. Hospitals able to provide at least 80% of pediatric patients with an operating room start time within 18 hours of admission to the emergency department score better as part of the overall pediatric orthopaedic ranking. Therefore, it is important to determine whether the 18-hour treatment time for pediatric femur shaft fractures is a clinically meaningful metric. Methods: A retrospective review of clinical outcomes of 174 pediatric patients (aged below 16 y) with isolated femur shaft fractures (Injury Severity Score=9) was conducted from 1997 to 2017 at a single level I pediatric trauma center. The 2 comparison groups were patients receiving fracture reduction within 18 hours of emergency department admission (N=87) or >18 hours (N=87). Results: Patient, injury, and surgical characteristics were similar between the 2 groups. Both groups had a similar mean age (treatment <18 h=7.5 y; treatment >18 h=8.1 y). Patients who received treatment within 18 hours were more often immobilized postoperatively (70.1% vs. 53.5%; P=0.0362) and had a shorter median hospital length of stay (2 vs. 3 d; P=0.0047). There were no statistically significant differences in any outcomes including surgical site infection, time to weight-bearing (treatment <18 h mean=48.1 d vs. 52.5 d), time to complete radiographic fracture healing (treatment <18 h mean=258.9 d vs. 232.0 d), decreased range of motion, genu varus/valgus, limb length discrepancy, loss of reduction, or persistent pain. Conclusions: Treatment of pediatric femur shaft fractures within 18 hours does not impact clinical outcomes. National quality measures should therefore use evidence-based metrics to help improve the standard of care. Level of Evidence: Therapeutic level III.

Abstract INTRODUCTION: Hemophagocytic lymphohistiocytosis (HLH) is a rare disorder caused by the pathologic activation of the immune system. In children, either a molecular diagnosis consistent with HLH or five out of the following eight criteria are considered necessary for a diagnosis of HLH (HLH-04 criteria): 1) fever; 2) splenomegaly; 3) cytopenia in two or more cell lines; 4) hypertriglyceridemia (≥265 mg/dL) or hypofibrinogenemia (≤150 mg/dL); 5) hemophagocytosis in the bone marrow, spleen, or lymph nodes; 6) hyperferritinemia (≥500 mcg/L); 7) impaired NK cell function; and 8) elevated soluble CD25 (sCD25). These criteria have been extrapolated to diagnose HLH in adults; however, it's unclear if these same criteria are applicable in the adult population. METHODS: We reviewed the Mayo Clinic electronic medical record for all adult (≥18 years) hospitalized patients with an admission serum ferritin of ≥500 mcg/L from January 2012 through December 2014. Patients' charts were reviewed and those who met the HLH-04 criteria were considered to have HLH. For the remainder of the patients, the etiology for hyperferritinemia was determined based on chart review and discharge diagnoses. Logistic regression models were used to assess the ability of these values in predicting a diagnosis of HLH. The Mayo Clinic IRB approved this study. RESULTS: We identified 1,329 patients with a serum ferritin ≥500 mcg/L. Of these, HLH was diagnosed in 28 (2.1%) patients (malignancy-associated HLH in 11 patients, infection-associated HLH in 4 patients, autoimmune-associated HLH in 7 patients, and idiopathic HLH in 6 patients). Table 1 describes the etiology of hyperferritinemia in the remaining 1,301 patients. In contrast to pediatric hospitalized patients (Allen, Pediatr Blood Cancer, 2008), adults are more likely to have malignancy (28.1% vs 7%; p<0.05); bacterial infection (21% vs. 13%; p=0.001); liver disease (9.9% vs. 2.7%; p<0.05); and cardiac disease (7.2% vs. 1.5%; p=0.0001) as the etiology of hyperferritinemia during their hospitalization. Among all patients in the study, the following variables were associated with higher odds of having HLH compared to hyperferritinemia due to an alternate cause: elevated ferritin (odds ratio [OR] 35.97, p<0.01); thrombocytopenia (OR 15.22, p<0.01), cytopenias defined by HLH-04 criteria (OR 8.04, p<0.01); elevated admission serum bilirubin (OR 1.05, p=0.02); and peak serum bilirubin (OR 1.05, p<0.01). After stepwise selection in multivariate analysis, serum ferritin ≥2,600 mcg/L and platelets ≤100 x 109/L were independently associated with HLH diagnosis (OR 24.9 and 7.8 respectively; p<0.01 for both). The area under the curve (AUC or c-statistic) ranges from 0.5 for no ability to discriminate, to 1.0 for perfect discrimination; this model has an AUC of 0.91, which shows very good discrimination between cases and controls. An adult hospitalized patient with a combination of serum ferritin ≥2,600 mcg/L and platelets ≤100x109/L predicts a ~200 fold increased likelihood of being diagnosed with HLH as compared to hospitalized adult meeting neither of these criteria (Figures 1 and 2). CONCLUSION: In contrast to hospitalized pediatric patients, the etiology of hyperferritinemia in adults is more likely to be malignancy, bacterial infection, liver disease, and cardiac disease. We conclude that a combination of serum ferritin ≥2,600 mcg/L and platelet count ≤100 x 109/L can be used as screening criteria to help identify adult patients most likely to have HLH. The traditional 5/8 criteria to diagnose HLH in pediatric patients do not appear necessary for establishing a diagnosis of HLH in adult patients. These observations need replication in an independent data set prior to broader applicability. Table 1.Patients with ferritin > 500 mcg/L with underlying cause for elevation.Number Diagnosed (%)Cardiac Disease95 (7.2%)Liver Disease131 (9.9%)Renal Disease87 (6.6%)Infectious343 (25.8%)Malignancy373 (28.1%)Autoimmune118 (8.9%)Solid Organ Transplant34 (2.6%)Stem Cell Transplant16 (1.2%)Bone Marrow Failure9 (0.7%)Shock39 (2.9%)Idiopathic53 (4.0%)Hemoglobinopathies3 (0.2%) Figure 1. ROC curve for the multivariable model with ferritin > 2,600 (yes/no) and platelets below 100,000 (yes/no) in the model with a very good discrimination c-statistic of 0.91. Figure 1. ROC curve for the multivariable model with ferritin > 2,600 (yes/no) and platelets below 100,000 (yes/no) in the model with a very good discrimination c-statistic of 0.91. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

Abstract We and others previously demonstrated an increased risk of monoclonal gammopathy of undetermined significance (MGUS) in first-degree relatives of multiple myeloma (MM) and MGUS patients, underscoring a familial component to this disease. However, MM is known to be a collection of cytogenetically distinct diseases, with 40-50% of MM cases having a reciprocal translocation involving the IgH locus on chromosome 14q32 and in many of the remaining patients, trisomies. In a small proportion of cases, both IgH abnormalities and trisomies are found in the same clone. These MM cytogenetic subtypes are associated with differential prognosis. Whether there are differences in familial risk by cytogenetic subtypes is not known. Patients with MM were recruited from the Mayo Clinic Hematology practice in Rochester, Minnesota (MN) between 2005 and 2015. Serum samples were collected from first-degree relatives and subjected to electrophoresis and immunofixation for MGUS determination. Age- and sex-specific rates of MGUS from the population-based Olmsted County, MN, prevalence study were used as expected rates for the calculation of risk ratios (RRs) for comparisons of first-degree relatives. Cytogenetic subtypes were classified by FISH testing performed in the clinic and categorized into groups defined by IgH translocation , trisomy, intermediate/ high-risk disease (t(4;14), t(14;16), t(14;20), and del17p). Further, the distribution of cytogenetic subtypes was compared between MM cases with and without evidence of MGUS in first degree relatives using Chi square tests. Analyses were also subset to MM probands diagnosed in MN. There were a total of 442 MM probands, with 1235 first-degree relatives recruited and eligible for the study. MGUS was detected in 78 relatives, for an age- and sex-adjusted prevalence of 6.1% (4.7%-7.5%). Analyses comparing prevalence of MGUS in first degree relatives to population-based rates from Olmsted County showed a 2.5-fold (95% CI=1.9-3.0) increased prevalence. When performing analyses among MN probands only (RR=2.9; 95% CI=2.1-3.8), results were similar. Of the 442 probands, 310 had FISH performed on plasma cells (identified by cIG staining; cIg-FISH) at diagnosis. Of these cases, 49% had a trisomy, 23% had an IgH translocation, and 9% had both. Although the proportion of trisomies and IgH translocations were 4-6% higher among MM probands with a relative with MGUS compared to those without, these differences were not statistically significant (Table 1). MM probands with a first degree relative with MGUS also had a similar proportion of cytogenetic subtypes that are considered of intermediate or high risk compared to those MM without a family history (26.9% vs 25.2%) (p=0.79) (Table 1). When restricted to MM probands from MN, results remained non-statistically significant (Table 1). We showed an increased risk of MGUS in first-degree relatives of MM cases, implying shared environment and/or genetics underlying this malignancy. However, we did not find an association between family history of MGUS in MM probands and the cytogenetic subtypes examined. Larger cohorts are needed to examine if a relationship exists between cytogenetic subtypes of MM and familial risk. Disclosures Kumar: Noxxon Pharma: Consultancy, Research Funding; Array BioPharma: Consultancy, Research Funding; AbbVie: Research Funding; Onyx: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Glycomimetics: Consultancy; Kesios: Consultancy; Janssen: Consultancy, Research Funding; Skyline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Research Funding; BMS: Consultancy.

Background: Spontaneous coronary artery dissection (SCAD) is an uncommon idiopathic disorder predominantly affecting young, otherwise healthy women. Rare familial cases reveal a genetic predisposition to disease. The aim of this study was to identify a novel susceptibility gene for SCAD. Methods: Whole-exome sequencing was performed in a family comprised of 3 affected individuals and filtered to identify rare, predicted deleterious, segregating variants. Immunohistochemical staining was used to evaluate protein expression of the identified candidate gene. The prevalence and spectrum of rare (<0.1%) variants within binding domains was determined by next-generation sequencing or denaturing high-performance liquid chromatography in a sporadic SCAD cohort of 675 unrelated individuals. Results: We identified a rare heterozygous missense variant within a highly conserved β-integrin–binding domain of TLN1 segregating with familial SCAD. TLN1 encodes talin 1—a large cytoplasmic protein of the integrin adhesion complex that links the actin cytoskeleton and extracellular matrix. Consistent with high mRNA expression in arterial tissues, robust immunohistochemical staining of talin 1 was demonstrated in coronary arteries. Nine additional rare heterozygous missense variants in TLN1 were identified in 10 sporadic cases. Incomplete penetrance, suggesting genetic or environmental modifiers of this episodic disorder, was evident in the familial case and 5 individuals with sporadic SCAD from whom parental DNA was available. Conclusions: Our findings reveal TLN1 as a disease-associated gene in familial and sporadic SCAD and, together with abnormal vascular phenotypes reported in animal models of talin 1 disruption, implicate impaired structural integrity of the coronary artery cytoskeleton in SCAD susceptibility.

AbstractHigher ratios of the pre‐treatment peripheral blood absolute lymphocyte (ALC) to absolute monocyte counts (AMC) are associated with improved outcomes in lymphoma. Conversely, elevated pre‐treatment serum cytokines are associated with inferior outcomes. The relationship between pre‐treatment serum cytokines and ALC/AMC ratios remains unknown. We studied twelve serum cytokines and the ALC/AMC ratios in 390 patients with untreated diffuse large B‐cell, follicular, mantle cell, T‐cell, and Hodgkin lymphoma. Different pre‐treatment serum cytokine concentrations correlated with ALC, AMC, and ALC/AMC ratios depending on the lymphoma type. In the entire cohort (n = 390) lower ALC/AMC ratios modestly correlated with higher IL‐2R (r = −0.36), IL‐12 (r = −0.17), IP‐10 (r = −0.23), and MIG (r = −0.32) concentrations (p < 0.001). Elevated IL‐2R was independently associated with suppressed ALC (OR 2.69, 95% CI 1.77–4.07,p < 0.001), elevated AMC (OR 2.05, 95% CI 1.34–3.14,p < 0.001), and suppressed ALC/AMC ratios (OR 3.51, 95% CI 2.31–5.34,p < 0.001). Both elevated IL‐2R (HR 2.27, 95% CI 1.48–3.49,p < 0.001) and suppressed ALC/AMC ratios (HR 1.53, 95% CI 1.03–2.28,p = 0.037) were independently associated with inferior overall survival. These data support the notion that elevated serum cytokines are immunosuppressive and provide further rationale to target the tumor microenvironment for therapeutic benefit.