Martin J. Hessner

Making a definitive diagnosis: Successful clinical application of whole exome sequencing in a child with intractable inflammatory bowel disease

Purpose: We report a male child who presented at 15 months with perianal abscesses and proctitis, progressing to transmural pancolitis with colocutaneous fistulae, consistent with a Crohn disease-like illness. The age and severity of the presentation suggested an underlying immune defect; however, despite comprehensive clinical evaluation, we were unable to arrive at a definitive diagnosis, thereby restricting clinical management.Methods: We sought to identify the causative mutation(s) through exome sequencing to provide the necessary additional information required for clinical management.Results: After sequencing, we identified 16,124 variants. Subsequent analysis identified a novel, hemizygous missense mutation in the X-linked inhibitor of apoptosis gene, substituting a tyrosine for a highly conserved and functionally important cysteine. X-linked inhibitor of apoptosis was not previously associated with Crohn disease but has a central role in the proinflammatory response and bacterial sensing through the NOD signaling pathway. The mutation was confirmed by Sanger sequencing in a licensed clinical laboratory. Functional assays demonstrated an increased susceptibility to activation-induced cell death and defective responsiveness to NOD2 ligands, consistent with loss of normal X-linked inhibitor of apoptosis protein function in apoptosis and NOD2 signaling.Conclusions: Based on this medical history, genetic and functional data, the child was diagnosed as having an X-linked inhibitor of apoptosis deficiency. Based on this finding, an allogeneic hematopoietic progenitor cell transplant was performed to prevent the development of life-threatening hemophagocytic lymphohistiocytosis, in concordance with the recommended treatment for X-linked inhibitor of apoptosis deficiency. At >42 days posttransplant, the child was able to eat and drink, and there has been no recurrence of gastrointestinal disease, suggesting this mutation also drove the gastrointestinal disease. This report describes the identification of a novel cause of inflammatory bowel disease. Equally importantly, it demonstrates the power of exome sequencing to render a molecular diagnosis in an individual patient in the setting of a novel disease, after all standard diagnoses were exhausted, and illustrates how this technology can be used in a clinical setting.

An Autoinflammatory Disease Due to Homozygous Deletion of the IL1RN Locus

We describe a patient with an autoinflammatory disease in which the main clinical features are pustular rash, marked osteopenia, lytic bone lesions, respiratory insufficiency, and thrombosis. Genetic studies revealed a 175-kb homozygous deletion at chromosome 2q13, which encompasses several interleukin-1 family members, including the gene encoding the interleukin-1-receptor antagonist (IL1RN). Mononuclear cells, obtained from the patient and cultured, produced large amounts of inflammatory cytokines, with increasing amounts secreted after stimulation with lipopolysaccharide. A similar increase was not observed in peripheral-blood mononuclear cells from a patient with neonatal-onset multisystem inflammatory disorder (NOMID). Treatment with anakinra completely resolved the symptoms and lesions.

Blockade of interleukin-6 signaling augments regulatory T-cell reconstitution and attenuates the severity of graft-versus-host disease.

Graft-versus-host disease (GVHD) is the major complication after allogeneic bone marrow transplantation and is characterized by the overproduction of proinflammatory cytokines. In this study, we have identified interleukin-6 (IL-6) as a critical inflammatory cytokine that alters the balance between the effector and regulatory arms of the immune system and drives a proinflammatory phenotype that is a defining characteristic of GVHD. Our results demonstrate that inhibition of the IL-6 signaling pathway by way of antibody-mediated blockade of the IL-6 receptor (IL-6R) markedly reduces pathologic damage attributable to GVHD. This is accompanied by a significant increase in the absolute number of regulatory T cells (Tregs) that is due to augmentation of thymic-dependent and thymic-independent Treg production. Correspondingly, there is a significant reduction in the number of T helper 1 and T helper 17 cells in GVHD target organs, demonstrating that blockade of IL-6 signaling decreases the ratio of proinflammatory T cells to Tregs. These studies demonstrate that antibody blockade of the IL-6R serves to recalibrate the effector and regulatory arms of the immune system and represents a novel, potentially clinically translatable, strategy for the attenuation of GVHD.

Rapid determination of platelet alloantigen genotypes by polymerase chain reaction using allele-specific primers

BACKGROUND: The technique of allele‐specific polymerase chain reaction (PCR) amplification has been adapted for DNA‐based human platelet alloantigen typing.

Identification of a Molecular Signature in Human Type 1 Diabetes Mellitus Using Serum and Functional Genomics

Understanding active proinflammatory mechanisms at and before type 1 diabetes mellitus (T1DM) onset is hindered in humans, given that the relevant tissues are inaccessible and pancreatic immune responses are difficult to measure in the periphery by traditional approaches. Therefore, we investigated the use of a sensitive and comprehensive genomics strategy to investigate the presence of proinflammatory factors in serum. The sera of recent onset diabetes patients (n = 15, 12 possessing and 3 lacking islet cell autoantibodies), long-standing diabetes patients (n = 12), “at risk” siblings of diabetes patients (n = 9), and healthy controls (n = 12) were used to induce gene expression in unrelated, healthy PBMC. After culture, gene expression was measured with microarrays and normalized expression data were subjected to hierarchical clustering and multidimensional scaling. All recent onset sera induced an expression signature (192 UniGenes; fold change: >1.5, p < 0.01; false discovery rate: <0.01) that included IL-1 cytokine family members and chemokines involved in monocyte/macrophage and neutrophil chemotaxis, as well as numerous receptors and signaling molecules. This molecular signature was not induced with the sera of healthy controls or long standing diabetes patients, where longitudinal analysis of “at risk” siblings (n = 3) before and after onset support the hypothesis that the signature emerges years before onset. This study supports prior investigations of serum that reflect disease processes associated with progression to T1DM. Identification of unique inflammatory mediators may improve disease prediction beyond current islet autoantibodies. Furthermore, proinflammatory serum markers may be used as inclusion criteria or endpoint measures in clinical trials aimed at preventing T1DM.

Determination of neutrophil antigen gene frequencies in five ethnic groups by polymerase chain reaction with sequence-specific primers

Background: The granulocyte antigens NA1 and NA2 are the two recognized allelic forms of Fcγ receptor IIIB. These antigens are clinically relevant, because they are the most frequent targets of neutrophil antibodies in alloimmune neonatal neutropenia, transfusion‐related acute lung injury, and chronic benign autoimmune neutropenia of infancy.

Activated protein C targets CD8+ dendritic cells to reduce the mortality of endotoxemia in mice

Activated protein C (aPC) therapy reduces mortality in adult patients with severe sepsis. In mouse endotoxemia and sepsis models, mortality reduction requires the cell signaling function of aPC, mediated through protease-activated receptor-1 (PAR1) and endothelial protein C receptor (EPCR; also known as Procr). Candidate cellular targets of aPC include vascular endothelial cells and leukocytes. Here, we show that expression of EPCR and PAR1 on hematopoietic cells is required in mice for an aPC variant that mediates full cell signaling activity but only minimal anticoagulant function (5A-aPC) to reduce the mortality of endotoxemia. Expression of EPCR in mature murine immune cells was limited to a subset of CD8+ conventional dendritic cells. Adoptive transfer of splenic CD11chiPDCA-1- dendritic cells from wild-type mice into animals with hematopoietic EPCR deficiency restored the therapeutic efficacy of aPC, whereas transfer of EPCR-deficient CD11chi dendritic cells or wild-type CD11chi dendritic cells depleted of EPCR+ cells did not. In addition, 5A-aPC inhibited the inflammatory response of conventional dendritic cells independent of EPCR and suppressed IFN-gamma production by natural killer-like dendritic cells. These data reveal an essential role for EPCR and PAR1 on hematopoietic cells, identify EPCR-expressing dendritic immune cells as a critical target of aPC therapy, and document EPCR-independent antiinflammatory effects of aPC on innate immune cells.

Quantitative quality control in microarray experiments and the application in data filtering, normalization and false positive rate prediction

Data preprocessing including proper normalization and adequate quality control before complex data mining is crucial for studies using the cDNA microarray technology. We have developed a simple procedure that integrates data filtering and normalization with quantitative quality control of microarray experiments. Previously we have shown that data variability in a microarray experiment can be very well captured by a quality score q(com) that is defined for every spot, and the ratio distribution depends on q(com). Utilizing this knowledge, our data-filtering scheme allows the investigator to decide on the filtering stringency according to desired data variability, and our normalization procedure corrects the q(com)-dependent dye biases in terms of both the location and the spread of the ratio distribution. In addition, we propose a statistical model for false positive rate determination based on the design and the quality of a microarray experiment. The model predicts that a lower limit of 0.5 for the replicate concordance rate is needed in order to be certain of true positives. Our work demonstrates the importance and advantages of having a quantitative quality control scheme for microarrays.

T cell-depleted allogeneic bone marrow transplantation for high-risk non-Hodgkin's lymphoma : clinical and molecular follow-up

The use of allogeneic BMT in patients with relapsed non-Hodgkin lymphoma (NHL) offers the advantage of tumor-free bone marrow and possibly a ‘graft-versus-lymphoma effect’ which may decrease the risk of recurrence. However, allogeneic BMT also poses an increased risk of death due to graft-versus-host disease (GVHD) which can be ameliorated by T cell depletion. We performed a retrospective review of 37 patients who underwent T cell-depleted allogeneic BMT for aggressive and indolent NHL between 1988 and 1996. Polymerase chain reaction (PCR) was used to identify indolent NHL patients with the BCL2/IgH translocation which served as a marker of residual disease. Sixteen of 37 patients (44%) are alive and progression-free with a median follow-up of 4.4 years (range 1–10.3). The incidence of grade 2–4 acute GVHD was 36% and extensive chronic GVHD developed in 12%. Patients with aggressive NHL have an overall PFS of 33% (12–54%); those with chemotherapy-resistant and sensitive disease have PFS of 17% (0–47%), and 40% (15–65%) respectively at 5 years. Patients with indolent histologies have overall PFS of 62% (37–86%); those with chemotherapy-resistant and sensitive disease have PFS of 55% (25–85%) and 80% (45–100%) respectively at 5 years. Eight patients with indolent disease had a BCL2/IgH translocation detectable by PCR. Five of these eight patients remain alive and progression free at a median of 6.5 years after BMT (range 2.1–7.4 years), four of whom remain PCR positive from 1.7 to 2.9 years after transplantation. We conclude that T cell-depleted allogeneic BMT poses a low risk for death due to GVHD, and should be considered for patients with relapsed and refractory indolent NHL.

Apoptosis of CD4+CD25high T Cells in Type 1 Diabetes May Be Partially Mediated by IL-2 Deprivation

Background Type 1 diabetes (T1D) is a T-cell mediated autoimmune disease targeting the insulin-producing pancreatic β cells. Naturally occurring FOXP3+CD4+CD25high regulatory T cells (Tregs) play an important role in dominant tolerance, suppressing autoreactive CD4+ effector T cell activity. Previously, in both recent-onset T1D patients and β cell antibody-positive at-risk individuals, we observed increased apoptosis and decreased function of polyclonal Tregs in the periphery. Our objective here was to elucidate the genes and signaling pathways triggering apoptosis in Tregs from T1D subjects. Principal Findings Gene expression profiles of unstimulated Tregs from recent-onset T1D (n = 12) and healthy control subjects (n = 15) were generated. Statistical analysis was performed using a Bayesian approach that is highly efficient in determining differentially expressed genes with low number of replicate samples in each of the two phenotypic groups. Microarray analysis showed that several cytokine/chemokine receptor genes, HLA genes, GIMAP family genes and cell adhesion genes were downregulated in Tregs from T1D subjects, relative to control subjects. Several downstream target genes of the AKT and p53 pathways were also upregulated in T1D subjects, relative to controls. Further, expression signatures and increased apoptosis in Tregs from T1D subjects partially mirrored the response of healthy Tregs under conditions of IL-2 deprivation. CD4+ effector T-cells from T1D subjects showed a marked reduction in IL-2 secretion. This could indicate that prior to and during the onset of disease, Tregs in T1D may be caught up in a relatively deficient cytokine milieu. Conclusions In summary, expression signatures in Tregs from T1D subjects reflect a cellular response that leads to increased sensitivity to apoptosis, partially due to cytokine deprivation. Further characterization of these signaling cascades should enable the detection of genes that can be targeted for restoring Treg function in subjects predisposed to T1D.