Yves Pastore

Improving diagnostic precision, care and syndrome definitions using comprehensive next-generation sequencing for the inherited bone marrow failure syndromes

Background Phenotypic overlap among the inherited bone marrow failure syndromes (IBMFSs) frequently limits the ability to establish a diagnosis based solely on clinical features. >70 IBMFS genes have been identified, which often renders genetic testing prolonged and costly. Since correct diagnosis, treatment and cancer surveillance often depend on identifying the mutated gene, strategies that enable timely genotyping are essential. Methods To overcome these challenges, we developed a next-generation sequencing assay to analyse a panel of 72 known IBMFS genes. Cases fulfilling the clinical diagnostic criteria of an IBMFS but without identified causal genotypes were included. Results The assay was validated by detecting 52 variants previously found by Sanger sequencing. A total of 158 patients with unknown mutations were studied. Of 75 patients with known IBMFS categories (eg, Fanconi anaemia), 59% had causal mutations. Among 83 patients with unclassified IBMFSs, we found causal mutations and established the diagnosis in 18% of the patients. The assay detected mutant genes that had not previously been reported to be associated with the patient phenotypes. In other cases, the assay led to amendments of diagnoses. In 20% of genotype cases, the results indicated a cancer surveillance programme. Conclusions The novel assay is efficient, accurate and has a major impact on patient care.

A Transgenic Mouse Model of Autoimmune Glomerulonephritis and Necrotizing Arteritis Associated with Cryoglobulinemia

Mice implanted with hybridoma secreting 6-19 IgG3 anti-IgG2a rheumatoid factor (RF) with cryoglobulin activity develop acute glomerulonephritis and cutaneous leukocytoclastic vasculitis. As the RF activity is implicated in the skin, but not glomerular lesions, it is still unclear whether the renal pathogenicity is determined by 6-19 H chains alone or their combination with L chains. To address this question, we have generated transgenic mice expressing only the H chain gene or both H and L chain genes of the 6-19 IgG3 anti-IgG2a RF and determined the development of glomerular and vascular lesions. H-single and H/L-double transgenic mice displayed comparable high amounts of IgG3 cryoglobulins, but only H/L-double transgenic mice having 10-fold higher levels of IgG3 anti-IgG2a RF progressively developed chronic, lethal glomerulonephritis. The severe glomerular lesions observed at 8–10 mo of age were very heterogeneous (membranoproliferative changes, crescents, and sclerosis); in addition, one-third of them had necrotizing arteritis in the kidneys and skeletal muscles. These renal and vascular changes were very different from those observed in the acute cryoglobulinemia, characterized by mainly “wire-loop” glomerular lesions and a cutaneous leukocytoclastic form of vasculitis. Thus, our data demonstrate the importance of a unique combination of the H and L chains for the expression of the pathogenic activity of IgG3 cryoglobulins and that a single autoantibody is able to induce different types of glomerular and vascular complications, depending on its production levels and kinetics.

Thrombopoietin Receptor Agonist Use in Children: Data From the Pediatric ITP Consortium of North America ICON2 Study

Data on second‐line treatment options for pediatric patients with immune thrombocytopenia (ITP) are limited. Thrombopoietin receptor agonists (TPO‐RA) provide a nonimmunosuppressive option for children who require an increased platelet count.

Role of neutrophils in murine cryoglobulinemia

Abstract. Murine IgG3 anti-IgG2a rheumatoid factor (RF) monoclonal antibodies (mAb) with cryoglobulin activity, are able to induce, in normal mice, skin leukocytoclastic vasculitis and lupus-like glomerulonephritis resembling ‘wire-loop’ lesions (subendothelial immune deposits). The development of glomerular, but not skin, lesions in immunoglobulin-deficient mice (lacking the corresponding IgG2a autoantigen) receiving IgG3 RF cryoglobulins indicates that the RF activity of IgG3 monoclonal cryoglobulins and subsequent formation of IgG3-IgG2a immune complexes play a critical role in the development of skin vasculitis. In contrast, nephritogenic activity is solely contributed by IgG3-associated cryoglobulin activity. Polymorphonuclear leukocyte (PMN) infiltration is one of the major pathologic changes observed in both types of lesions. Treatment with mAbs against the adhesion molecules leukocyte function-associated antigen 1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1) (both known for their involvement in PMN-endothelial cell interaction) inhibits the development of skin vascular lesions. However, it has no effect on the generation of glomerulonephritis. Apparently, adhesion molecule requirements for PMN interaction with glomerular capillary endothelial cells are different from those for PMN infiltration of the skin. However, the PMN depletion experiment has clearly shown that PMNs play an active role in the development of ‘wire-loop’ glomerular lesions. In the absence of the glomerular PMN infiltration, IgG3 RF cryoglobulins induce a different type of glomerular lesion, characterized by voluminous intracapillary thrombi and mesangial deposits, yet lacking subendothelial deposits. This is consistent with the fact that the latter lesions can be induced by certain IgG3 mAbs, which are unable to provoke glomerular PMN infiltration. Finally, the activation of the complement system does not appear to play a major role in either skin or glomerular lesions induced by IgG3 RF cryoglobulins.

The impact of category, cytopathology and cytogenetics on development and progression of clonal and malignant myeloid transformation in inherited bone marrow failure syndromes

Inherited bone marrow failure syndromes are a group of rare, heterogeneous genetic disorders with a risk of clonal and malignant myeloid transformation including clonal marrow cytogenetic abnormalities, myelodysplastic syndrome and acute myeloid leukemia. The clinical characteristics, risk classification, prognostic factors and outcome of clonal and malignant myeloid transformation associated with inherited bone marrow failure syndromes are largely unknown. The aims of this study were to determine the impact of category, cytopathology and cytogenetics, the three components of the “Category Cytology Cytogenetics” classification of pediatric myelodysplastic syndrome, on the outcome of clonal and malignant myeloid transformation associated with inherited bone marrow failure. We used data from the Canadian Inherited Marrow Failure Registry. Among 327 patients with inherited bone marrow failure syndrome enrolled in the registry, the estimated risk of clonal and malignant myeloid transformation by the age of 18 years was 37%. The risk of clonal and malignant myeloid transformation varied according to the type of inherited bone marrow failure syndrome but was highest in Fanconi anemia. The development of clonal and malignant myeloid transformation significantly affected overall survival. Mortality varied based on cytopathological group. The largest group of patients had refractory cytopenia. Clonal marrow cytogenetic abnormalities were identified in 87% of patients with clonal and malignant myeloid transformation, and different cytogenetic groups had different impacts on disease progression. We conclude that category, cytopathology and cytogenetics in cases of clonal and malignant myeloid transformation associated with inherited bone marrow failure syndromes have an important impact on outcome and that the classification of such cases should incorporate these factors.

Airway hyperreactivity is frequent in non‐asthmatic children with sickle cell disease

Asthma is associated with poorer outcomes in sickle cell disease (SCD). Whether AHR can exist in SCD as a distinct entity, separate and independent of asthma, is unknown.

Intravenous immunoglobulin–associated thrombosis: is it such a rare event? Report of a pediatric case and of the Quebec Hemovigilance System

Intravenous immunoglobulin (IVIG) is frequently given in autoimmune disorders. Side effects are usually mild but severe complications such as thrombosis may occur. After one patient with IVIG‐associated thrombotic complication at Sainte‐Justine Hospital, the incidence of serious adverse events (SAEs) reported to the Quebec Hemovigilance System (QHS) was reviewed.

An experimental model of cryoglobulin-associated vasculitis in mice

Cryoglobulins are immunoglobulins (Ig) that precipitate at temperatures lower than 37~ with resolution upon warming [1-3]. Since their initial characterization by Lerner et al. [1 ], it has been recognized that they are not a single distinct group of Ig, but heterogeneous with respect to their composition and physical properties. Cryoglobulins can be classified into three types [3] according to the presence in the cryoprecipitates of: monoclonal lg (type I), both monoclonal and polyclonal Ig (type II), or polyclonal ig with or without other serum proteins or exogenous antigens (type III). Monoclonal type I cryoglobulins are mostly associated with various lymphoproliferative disorders. Mixed type 1I or type III cryoglobulins are often found in serum from patients with atitoimmune diseases such as systemic hipus erythematosus and rheumatoid arthritis or with a variety of chronic infectious diseases. Clearly, the interaction between Ig molecules plays a critical role in the formation of cryoglobulins, since the sole or major components of a vast maiority of cryoglobulins are lg. The demonstration of rheumatoid factor (RF) and/or anti-idiotypic-like activity in IgM present in mixed cryoglobulins suggests the involvement of immune mechanisms in the generation of mixed cryoglobulins [2-6]. However, one should be aware that mixed cryoglobulins could be generated following in vitro, but not in vivo, interaction of low-affinity IgM RF with IgG complexes during the incubation of sera in test tubes at 4~ to isolate cryoglobulins. The molecular mechanisms responsible for the generation of type 1 monoclonal cryoglobulins are less clear. Available data suggest that several different mechanisms may be operative depending upori the particular cryoimmunoglobulins. Certain monoclonal Ig appear to form cryoprecipitates by self-association of Ig molecules via nonspecific physicochemical interaction [7-9], although a common physicochemical mechanism underlying such interactions has not been elucidated. An alternative, intriguing hypothesis is that cryoglobulins represent temperature-sensitive immunological interactions between the Fab region of the cryoprotein and antigenic determinants on the same molecules, i.e. an autoantibody-like reaction, as shown in

Clinical spectrum of pyruvate kinase deficiency: Data from the pyruvate kinase deficiency natural history study

An international, multicenter registry was established to collect retrospective and prospective clinical data on patients with pyruvate kinase (PK) deficiency, the most common glycolytic defect causing congenital nonspherocytic hemolytic anemia. Medical history and laboratory and radiologic data were retrospectively collected at enrollment for 254 patients with molecularly confirmed PK deficiency. Perinatal complications were common, including anemia that required transfusions, hyperbilirubinemia, hydrops, and prematurity. Nearly all newborns were treated with phototherapy (93%), and many were treated with exchange transfusions (46%). Children age 5 years and younger were often transfused until splenectomy. Splenectomy (150 [59%] of 254 patients) was associated with a median increase in hemoglobin of 1.6 g/dL and a decreased transfusion burden in 90% of patients. Predictors of a response to splenectomy included higher presplenectomy hemoglobin (P = .007), lower indirect bilirubin (P = .005), and missense PKLR mutations (P = .0017). Postsplenectomy thrombosis was reported in 11% of patients. The most frequent complications included iron overload (48%) and gallstones (45%), but other complications such as aplastic crises, osteopenia/bone fragility, extramedullary hematopoiesis, postsplenectomy sepsis, pulmonary hypertension, and leg ulcers were not uncommon. Overall, 87 (34%) of 254 patients had both a splenectomy and cholecystectomy. In those who had a splenectomy without simultaneous cholecystectomy, 48% later required a cholecystectomy. Although the risk of complications increases with severity of anemia and a genotype-phenotype relationship was observed, complications were common in all patients with PK deficiency. Diagnostic testing for PK deficiency should be considered in patients with apparent congenital hemolytic anemia and close monitoring for iron overload, gallstones, and other complications is needed regardless of baseline hemoglobin. This trial was registered at www.clinicaltrials.gov as #NCT02053480.

Gfi1b controls integrin signaling-dependent cytoskeleton dynamics and organization in megakaryocytes.

Mutations in GFI1B are associated with inherited bleeding disorders called GFI1B-related thrombocytopenias. We show here that mice with a megakaryocyte-specific Gfi1b deletion exhibit a macrothrombocytopenic phenotype along a megakaryocytic dysplasia reminiscent of GFI1B-related thrombocytopenia. GFI1B deficiency increases megakaryocyte proliferation and affects their ploidy, but also abrogates their responsiveness towards integrin signaling and their ability to spread and reorganize their cytoskeleton. Gfi1b-null megakaryocytes are also unable to form proplatelets, a process independent of integrin signaling. GFI1B-deficient megakaryocytes exhibit aberrant expression of several components of both the actin and microtubule cytoskeleton, with a dramatic reduction of α-tubulin. Inhibition of FAK or ROCK, both important for actin cytoskeleton organization and integrin signaling, only partially restored their response to integrin ligands, but the inhibition of PAK, a regulator of the actin cytoskeleton, completely rescued the responsiveness of Gfi1b-null megakaryocytes to ligands, but not their ability to form proplatelets. We conclude that Gfi1b controls major functions of megakaryocytes such as integrin-dependent cytoskeleton organization, spreading and migration through the regulation of PAK activity whereas the proplatelet formation defect in GFI1B-deficient megakaryocytes is due, at least partially, to an insufficient α-tubulin content.