Syndromes predisposing to leukemia are a major cause of inherited cytopenias in children.

Prolonged cytopenias are a non-specific sign with a wide differential diagnosis. Among inherited disorders, cytopenias predisposing to leukemia require a timely and accurate diagnosis to ensure appropriate medical management, including adequate monitoring and stem cell transplantation prior to the development of leukemia. We aimed to define the types and prevalences of the genetic causes leading to persistent cytopenias in children. The study comprises children with persistent cytopenias, myelodysplastic syndrome, aplastic anemia, or suspected inherited bone marrow failure syndromes, who were referred for genetic evaluation from all pediatric hematology centers in Israel during 2016-2019. For variant detection, we used Sanger sequencing of commonly mutated genes and a custom-made targeted next-generation sequencing panel covering 226 genes known to be mutated in inherited cytopenias; the minority subsequently underwent whole exome sequencing. In total, 189 children with persistent cytopenias underwent a genetic evaluation. Pathogenic and likely pathogenic variants were identified in 59 patients (31.2%), including 47 with leukemia predisposing syndromes. Most of the latter (32, 68.1%) had inherited bone marrow failure syndromes, nine (19.1%) had inherited thrombocytopenia predisposing to leukemia, and three each (6.4%) had predisposition to myelodysplastic syndrome or congenital neutropenia. Twelve patients had cytopenias with no known leukemia predisposition, including nine children with inherited thrombocytopenia and three with congenital neutropenia. In summary, almost one third of 189 children referred with persistent cytopenias had an underlying inherited disorder; 79.7% of whom had a germline predisposition to leukemia. Precise diagnosis of children with cytopenias should direct follow-up and management programs and may positively impact disease outcome.

Pediatric immune thrombocytopenia: apoptotic markers may help in predicting the disease course.

BACKGROUND: In all, 15-30% of pediatric immune thrombocytopenia (ITP) patients will remain chronically thrombocytopenic at 1 year post diagnosis. All attempts to classify patients at diagnosis have proven unsuccessful. We hypothesized that a different pathophysiology is responsible for non-chronic versus chronic pediatric ITP. We aimed to examine differences in the apoptotic markers' presentation at diagnosis between non-chronic and chronic patients. METHODS: Blood samples were collected from 42 pediatric patients with newly diagnosed ITP prior to initiation of treatment. We incubated patients' sera with control platelets and compared the results among three research groups: healthy controls, chronic ITP, and non-chronic ITP patients. We measured apoptotic markers phosphatidylserine (PS) exposure and mitochondrial inner membrane potential (ΔΨm) by flow cytometry and the level of human apoptotic proteins by Human Apoptosis Array. RESULTS: We found increased platelet PS exposure and decreased ΔΨm in response to all ITP patients' sera compared to control subjects. Human Apoptotic Array revealed an increased expression of five apoptotic proteins: BIM, CD40, IGFBP2, P21, and SMAC, following sera incubation of non-chronic pediatric ITP patients, compared to chronic patients' sera, at diagnosis. CONCLUSIONS: Our data contribute to knowledge on apoptosis markers that may aid in predicting the prognosis of children with ITP. IMPACT: The key message of our article is that children with chronic ITP have a different apoptotic profile compared to non-chronic ITP. Addition to existing literature: This is the first study comparing apoptotic markers between children with chronic ITP to non-chronic ITP. IMPACT: Our findings indicate that, in the future, apoptotic markers may help to classify ITP patients into non-chronic versus chronic ones, at diagnosis.

Quantitation of bleeding symptoms in a national registry of patients with inherited platelet disorders.

BACKGROUND: Inherited platelet deficiency and/or dysfunction may be more common in the general population than has previously been appreciated. In 2013 the Israeli Inherited Platelet Disorder (IPD) Registry was established. METHODS: Clinical and laboratory data were collected to pre-specified registration forms. The study protocol was approved by the local hospital ethics committees. RESULTS: To date we have included in the registry 89 patients (male 52%) from 79 families. Most patients (74%) have a not-yet specified inherited thrombocytopenia (n=39) or non-specific platelet function disorder (n=27). Full clinical data were available for 81 (91%) patients. The median (range) age at presentation and time of follow-up were 1.8years (1day-17.8years) and 4.7 (0-26) years, respectively. The Pediatric Bleeding Questionnaire was available for 78patients; abnormal bleeding score (≥2) was recorded in 47 (52.8%, 95% CI 42%-63.5%) patients and was less frequent in patients followed for isolated thrombocytopenia. Abnormal score was associated with a longer time of follow-up, OR 1.19 (95% CI 1.04-1.36). CONCLUSION: Long term follow-up of patients with IPDs is important as bleeding risks may increase with time. We expect that clinical and laboratory information of patients/families with IPDs gathered in a systemic format will allow for better diagnosis and treatment of these patients.

Persistent neonatal thrombocytopenia can be caused by IgA antiplatelet antibodies in breast milk of immune thrombocytopenic mothers.

Immune thrombocytopenia (ITP) in pregnant women can cause neonatal thrombocytopenia by transport of antiplatelet autoantibodies across the placenta. Usually, an infant's platelet count normalizes within 2 months. We observed neonatal thrombocytopenia that persisted more than 4 months and disappeared following discontinuation of breastfeeding. The aim of our study was to discern whether breast milk of ITP mothers contained antiplatelet antibodies causing persistent thrombocytopenia. We collected milk samples from 3 groups of women: ITP group, 7 women who had ITP during pregnancy; R-ITP group, 6 women who recovered from ITP before pregnancy; and 9 healthy controls. We found increased levels of antiplatelet antibodies of the immunoglobulin A type in the milk of ITP patients compared with the other 2 groups. Similar increase was demonstrated for antibodies binding to αIIbß3 expressed in cultured cells. Thus, transfer of antiplatelet antibodies from ITP mothers by breastfeeding can be associated with persistent neonatal thrombocytopenia.

Quantification of specific T and B cells immunological markers in children with chronic and transient ITP.

BACKGROUND: Immune thrombocytopenic purpura (ITP) is characterized by a transient (nonchronic) or permanent (chronic) decline in the number of platelets. Predicting the course of ITP, at the time of diagnosis, is of importance. Here we studied at diagnosis, clinical and immunological parameters in order to distinguish between different courses. The latter included the measure of new B and T cells using quantification of kappa-deleting recombination excision circles (KRECs) and T-cell receptor excision circles (TRECs), respectively. METHODS: Blood samples were collected from 44 children with a clinical diagnosis of ITP. Real-time PCR was performed in order to quantify the number of copies of TREC and KREC followed by collection of clinical data from medical files. The children were retrospectively divided into two groups: chronic and nonchronic. RESULTS: Twenty-four patients (54%) were classified as nonchronic ITP and 20 patients (46%) were classified as chronic ITP. We confirmed some clinical parameters (e.g., gender, age) but not others (e.g., preceding infection, level of thrombocytopenia) that distinguish patients with chronic and nonchronic course. While KREC quantification was similar in patients regardless the outcome of their disease, it was significantly higher than the level of controls (P < 0.05). TREC quantification was not different between patients and controls. CONCLUSIONS: KREC but not TREC levels are different in patients comparing to controls, pointing to an overreaction of B-cell development as a role in the pathogenesis of ITP. These results may shed more lights on the immune mechanism of ITP.

[DIVERSITY IN PRODUCT SELECTION AND THRESHOLDS FOR PLATELETS TRANSFUSION IN NEONATES AND PREMATURE INFANTS].

INTRODUCTION: Many premature and full-term newborns receive prophylactic platelet transfusions to prevent bleeding, particularly the most prevalent one, i.e, intracranial hemorrhages. However, the platelet count threshold above which bleeding is prevented and the efficacy of platelet transfusion in thrombocytopenic neonates, have yet to be established. Therefore, inter-Neonatal Intensive Care Units (NICU) variations in treatment indications and practices are expected. Considerable inter-NICU variations will emphasize the need for guidelines on platelet transfusions to neonates and premature infants. AIMS: To examine platelet products selection and indications for transfusion among neonatologists in Israel. Research and Methods: Electronic questionnaires addressing the choice of platelet products and the platelet count threshold for transfusion in various clinical settings were sent to 25 neonatal units. RESULTS: All 25 neonatal units responded (100% response rate). There was considerable variation in product selection among the different neonatal units. Up to 24% of the participating units reported selecting nontraditional products. Variation was also found in thresholds for platelet transfusion - several units used high thresholds while others used low ones. Traditional guidelines were followed in up to 64% of cases in selected clinical scenarios. CONCLUSIONS: There is considerable variation in both platelet product selection and platelet count thresholds for transfusion among the different neonatal units. DISCUSSION: A low threshold for platelet transfusion increases the risk for bleeding, whereas a high threshold increases the prevalence of complications from transfusion of blood products. Adherence to guidelines may prevent both such sequelae. Summary: Such variation in platelet transfusion among neonatologists emphasizes the need for an accepted policy. We recommend setting up a committee of neonatologists, pediatric hematologists and blood service experts which aims to establish an appropriate policy regarding the prevention of platelet transfusion sequelae in newborns.

Maternal and infantile hypercalcemia caused by vitamin-D-hydroxylase mutations and vitamin D intake.

BACKGROUND: Hypercalcemia is caused by many different conditions and may lead to severe complications. Loss-of-function mutations of CYP24A1, encoding vitamin D-24-hydroxylase, have recently been identified in idiopathic infantile hypercalcemia and in adult kidney stone disease. The aim of this study was to investigate the genetics and clinical features of both infantile and maternal hypercalcemia. METHODS: We studied members of four unrelated Israeli families with hypercalcemia, namely, one woman during pregnancy and after delivery and three infants. Clinical and biochemical data were obtained from probands' medical charts. Genomic DNA was isolated from peripheral blood and CYP24A1 was sequenced. RESULTS: Typical symptoms of hypercalcemia associated with the intake of recommended doses of vitamin D developed in the infants and pregnant woman. Four different loss-of-function CYP24A1 mutations were identified, two of which are reported here for the first time (p.Trp134Gly and p.Glu315*). The infants from families 1 and 2, respectively, were found to be compound heterozygotes, and the infant from family 3 and the pregnant woman were found to be homozygous. CONCLUSIONS: This is the first report of maternal hypercalcemia caused by a CYP24A1 mutation, showing that not only infants are at risk for this complication. Our findings emphasize the importance of recognition, genetic diagnosis and proper treatment of this recently identified hypercalcemic disorder in this era of widespread vitamin D supplements.

Hemophagocytic lymphohistiocytosis associated with parechovirus 3 infection.

Hemophagocytic lymphohistiocytosis (HLH) denotes the common final pathway of a potentially fatal hyperinflammatory condition of diverse etiologies. We describe the first case of documented HLH associated with human parechovirus 3. A monoallelic Ala91Val mutation was found in the PRF1 gene, but the contribution of this mutation to HLH remains controversial. The diagnosis, based on accepted criteria, was established early in the course of the disease and led to successful treatment and complete recovery. The awareness of this new association is clinically important in facilitating early treatment, preventing organ damage, and increasing the likelihood of complete recovery.

TCR-independent killing of B cell malignancies by anti-third-party CTLs: the critical role of MHC-CD8 engagement.

We previously demonstrated that anti-third-party CTLs (stimulated under IL-2 deprivation against cells with an MHC class I [MHC-I] background different from that of the host and the donor) are depleted of graft-versus-host reactivity and can eradicate B cell chronic lymphocytic leukemia cells in vitro or in an HU/SCID mouse model. We demonstrated in the current study that human allogeneic or autologous anti-third-party CTLs can also efficiently eradicate primary non-Hodgkin B cell lymphoma by inducing slow apoptosis of the pathological cells. Using MHC-I mutant cell line as target cells, which are unrecognizable by the CTL TCR, we demonstrated directly that this killing is TCR independent. Strikingly, this unique TCR-independent killing is induced through lymphoma MHC-I engagement. We further showed that this killing mechanism begins with durable conjugate formation between the CTLs and the tumor cells, through rapid binding of tumor ICAM-1 to the CTL LFA-1 molecule. This conjugation is followed by a slower second step of MHC-I-dependent apoptosis, requiring the binding of the MHC-I α2/3 C region on tumor cells to the CTL CD8 molecule for killing to ensue. By comparing CTL-mediated killing of Daudi lymphoma cells (lacking surface MHC-I expression) to Daudi cells with reconstituted surface MHC-I, we demonstrated directly for the first time to our knowledge, in vitro and in vivo, a novel role for MHC-I in the induction of lymphoma cell apoptosis by CTLs. Additionally, by using different knockout and transgenic strains, we further showed that mouse anti-third-party CTLs also kill lymphoma cells using similar unique TCR-independence mechanism as human CTLs, while sparing normal naive B cells.

Prenatal maternal stress predicts cord-blood ferritin concentration.

AIM: To examine the relationship between maternal stress in early pregnancy and cord-blood ferritin concentration. METHODS: The sample consisted of 140 pregnant women who lived in a region that was under rocket attack during a military operation (December 2008 to January 2009). Mothers in the stress group (n=63) were in their first trimester during this period. Mothers in the control group (n=77) became pregnant 4-5 months after the attacks ended. Maternal subjective stress was reported retrospectively. Cord-blood ferritin concentration was compared between stress and control groups, and was the dependent variable in a hierarchical multiple regression analysis. RESULTS: The mean cord-blood ferritin concentration was lower in the stress group compared to the control group (145.7±62.0 vs. 169.3±85.4 ng/mL, P<0.05). The cumulative distribution of cord-blood ferritin showed a shift to the left for the stress group. Hierarchical multiple regression analysis revealed that maternal subjective stress was a predictor for cord-blood ferritin concentration (hierarchical regression: ß=-0.18, P<0.05), especially in the stress group (simple slope analysis: ß=-0.32, P<0.01). CONCLUSION: Maternal stress during the first trimester of pregnancy is associated with lower cord-blood ferritin concentration.

Septicemia following rotavirus gastroenteritis.

BACKGROUND: Rotavirus gastroenteritis is a prevalent childhood illness rarely complicated by secondary bacterial sepsis. Although there are case reports of septicemia after rotavirus infection, there are no recent reviews on this topic. OBJECTIVES: To add new cases of septicemia after rotavirus to the literature, review the few cases of septicemia after rotavirus that have been reported, calculate the incidence of septicemia in children hospitalized for rotavirus gastroenteritis, and discuss the characteristics of septicemia after rotavirus infection and implications for current pediatric practice. METHODS: We identified children whose illness was complicated by septicemia from among all hospitalizations at our facility for rotavirus gastroenteritis from May 1999 through May 2010. We also review the few cases reported in the English literature. RESULTS: We identified two cases of septicemia from among 632 hospitalizations for rotavirus gastroenteritis in this time period, for an incidence rate of 0.32%, which is comparable to other estimates in the English literature. The typical course for cases of bacterial superinfection involves a second peak of high fever; other clinical signs are variable. CONCLUSIONS: Septicemia after rotavirus gastroenteritis is a rare but dangerous entity. Early identification of a child developing bacterial superinfection after rotavirus, as in any case of sepsis, is of the utmost importance, as is obtaining blood cultures in a child with a rotavirus infection and a second fever spike.

Spontaneous resolution of extreme thrombocytosis in 2 children.

Essential thrombocytosis (ET) is rare in children, sometimes difficult to be distinguished from secondary thrombocytosis. This report concerns 2 children with extreme thrombocytosis of 4100 × 10(9)/L and 1644 × 10(9)/L with partial and complete remission at 3 months and 4 years from diagnosis, with a follow-up of 4 and 17 years, respectively, with no cytoreduction therapy. Diagnosis of ET was suggested according to accepted criteria. However, spontaneous remission of the thrombocytosis argues for the diagnosis of secondary thrombocytosis. These patients highlight the complexity of distinguishing childhood ET from secondary thrombocytosis and the need for cautious personalized decision on cytoreduction therapy.

Reducing infantile anemia: insight on patterns of process and outcome indicators by ethnicity and socioeconomic class during a 10-year intervention program and 5 years after.

BACKGROUND: In 2005, Clalit Health Services (CHS), the largest health maintenance organization in Israel, initiated an intervention program aimed at reducing the prevalence rate of infantile anemia (IA). This study evaluated the progress made during the intervention (2005-2014) and its yield 5 years after it ended (2019). METHODS: The CHS database was retrospectively reviewed twice yearly from 2005 to 2014 for repetitive samples of children aged 9 to 18 months regarding the previous half-year interval, and a single sample in 2019. Data were collected on gender, ethnicity (Jewish/non-Jewish), socioeconomic class (SEC; low/intermediate/high), hemoglobin testing (yes/no), and hemoglobin level (if tested). Excluded were infants with documented or suspected hemoglobinopathy. RESULTS: At study initiation, the rate of performance of hemoglobin testing was 54.7%, and the IA prevalence rate was 7.8%. The performance rate was lower in the Jewish than the non-Jewish subpopulation. The low-SEC subpopulation had a similar hemoglobin testing rate to the high-SEC subpopulation but double the IA prevalence rate. Overall, by the end of the intervention (2014), the performance rate increased to 87.5%, and the AI prevalence rate decreased to 3.4%. In 2019, there was little change in the performance rate from the end of the intervention (88%) and the IA prevalence was further reduced to 2.7%. The non-Jewish and low-SEC subpopulations showed the most improvement which was maintained and even bettered 5 years after the intervention ended. CONCLUSIONS: The 10-year IA intervention program introduced by CHS in 2005 led to a reduction in IA prevalence rate to about 3.5% in all sub-populations evaluated. By program end, the results in the weaker subpopulations, which had the highest prevalence of IA at baseline, were not inferior to those in the stronger subpopulations. We recommended to the Israel Ministry of Health to adopt the intervention countrywide, and we challenge other countries to consider similar interventions.

Frequency and natural history of inherited bone marrow failure syndromes: the Israeli Inherited Bone Marrow Failure Registry.

BACKGROUND: Inherited bone marrow failure syndromes are rare genetic disorders characterized by bone marrow failure, congenital anomalies, and cancer predisposition. Available single disease registries provide reliable information regarding natural history, efficacy and side effects of treatments, and contribute to the discovery of the causative genes. However, these registries could not shed light on the true incidence of the various syndromes. We, therefore, established an Israeli national registry in order to investigate the relative frequency of each of these syndromes and their complications. DESIGN AND METHODS: Patients were registered by their hematologists in all 16 medical centers in Israel. We included patients with Fanconi anemia, severe congenital neutropenia, Diamond-Blackfan anemia, congenital amegakaryocytic thrombocytopenia, dyskeratosis congenita, Shwachman-Diamond syndrome, and thrombocytopenia with absent radii. RESULTS: One hundred and twenty-seven patients diagnosed between 1966 and 2007 were registered. Fifty-two percent were found to have Fanconi anemia, 17% severe congenital neutropenia, 14% Diamond-Blackfan anemia, 6% congenital amegakaryocytic thrombocytopenia, 5% dyskeratosis congenita, 2% Shwachman-Diamond syndrome, and 2% thrombocytopenia with absent radii. No specific diagnosis was made in only 2 patients. Of the thirty patients (24%) developing severe bone marrow failure, 80% had Fanconi anemia. Seven of 9 patients with leukemia had Fanconi anemia, as did all 6 with solid tumors. Thirty-four patients died from their disease; 25 (74%) had Fanconi anemia and 6 (17%) had severe congenital neutropenia. CONCLUSIONS: This is the first comprehensive population-based study evaluating the incidence and complications of the different inherited bone marrow failure syndromes. By far the most common disease was Fanconi anemia, followed by severe congenital neutropenia and Diamond-Blackfan anemia. Fanconi anemia carried the worst prognosis, with severe bone marrow failure and cancer susceptibility. Diamond-Blackfan anemia had the best prognosis. The data presented provide a rational basis for prevention programs and longitudinal surveillance of the complications of inherited bone marrow failure syndromes.

Type 2 Gaucher disease occurs in Ashkenazi Jews but is surprisingly rare.

Patients with Gaucher disease (GD) are divided into three types based on the presence and rate of progression of the neurologic manifestations. While type 1 GD has a strong predilection in the Jewish Ashkenazi population, both other types lack such a propensity. We report the occurrence of type 2 GD (GD2) in four pregnancies in two Jewish families in Israel (in one case the mother was not Ashkenazi but was from a Sfaradi Jewish family) and also review seven additional cases of GD2 in Ashkenazi Jewish families reported in the literature. Phenotypically, GD2 in Ashkenazi Jews does not differ significantly from this form in other ethnic groups. Genotypic analysis of probands from the two Israeli families demonstrates that each carried two heterozygous glucocerebrosidase mutations. We could find no explanation why GD2 is so rare in the Jewish Ashkenazi population but we could hypothesize that homozygosity for certain Ashkenazi alleles might be lethal, leading to a lower than expected frequency of GD2 and noted that no cases of homozygous L444P has ever been described in Ashkenazi Jews.

Langerhans cell histiocytosis in a premature baby presenting with skin-isolated disease: case report and literature review.

UNLABELLED: Langerhans cell histiocytosis (LCH) in premature babies is extremely rare as is a vesicular skin rash, while gastrointestinal involvement is associated with a poor outcome. We report a case of LCH in a premature baby presented with isolated vesiculo-papulo-macular skin lesions and insidiously developed gastrointestinal symptoms, haematological and severe pulmonary involvement. We also reviewed a few cases of LCH in premature babies in the English language medical literature. LCH in preterm babies appears to be a severe systemic disease, usually lethal in-utero or post delivery. CONCLUSION: Careful observation should be applied to newborns with skin-only Langerhans cell histiocytosis in order to identify in time progression to potentially fatal systemic disease.

Large-scale preparation of human anti-third-party veto cytotoxic T lymphocytes depleted of graft-versus-host reactivity: a new source for graft facilitating cells in bone marrow transplantation.

Induction of donor type chimerism in mildly prepared hosts without graft-versus-host disease (GvHD) is a most desirable goal in bone morrow transplantation. We have recently demonstrated in a mouse model that donor veto cytotoxic T lymphocytes (CTLs) can facilitate the induction of donor type chimerism in sublethally irradiated recipients without causing GvHD if they are effectively depleted of alloreactivity against host cells by means of stimulation against a third party. We extend this approach to human cells, by preparing CTLs in two major steps: primary culture in the absence of interleukin 2, leading to death by neglect of antihost clones, and addition of interleukin 2 and subsequent dilution of antihost clones as a consequence of the expansion of the anti-third-party clones. CTLs prepared in this way specifically suppress host cytotoxic T cells directed against antigens of the donor, but not against fourth-party antigens, as demonstrated in a standard (51)Cr release assay. We conclude that human anti-third-party CTLs afford a new source of veto cells that are depleted of potential graft-versus-host-reactive clones. The cells generated by this approach could potentially be used to facilitate engraftment of allogeneic hematopoietic stem cells.