Genetic backgrounds and clinical characteristics of congenital neutropenias in Israel.

BACKGROUND: Congenital neutropenias are characterized by severe infections and a high risk of myeloid transformation; the causative genes vary across ethnicities. The Israeli population is characterized by an ethnically diverse population with a high rate of consanguinity. OBJECTIVE: To evaluate the clinical and genetic spectrum of congenital neutropenias in Israel. METHODS: We included individuals with congenital neutropenias listed in the Israeli Inherited Bone Marrow Failure Registry. Sanger sequencing was performed for ELANE or G6PC3, and patients with wild-type ELANE/G6PC3 were referred for next-generation sequencing. RESULTS: Sixty-five patients with neutropenia were included. Of 51 patients with severe congenital neutropenia, 34 were genetically diagnosed, most commonly with variants in ELANE (15 patients). Nine patients had biallelic variants in G6PC3, all of consanguineous Muslim Arab origin. Other genes involved were SRP54, JAGN1, TAZ, and SLC37A4. Seven patients had cyclic neutropenia, all with pathogenic variants in ELANE, and seven had Shwachman-Diamond syndrome caused by biallelic SBDS variants. Eight patients (12%) developed myeloid transformation, including six patients with an unknown underlying genetic cause. Nineteen (29%) patients underwent hematopoietic stem cell transplantation, mostly due to insufficient response to treatment with granulocyte-colony stimulating factor or due to myeloid transformation. CONCLUSIONS: The genetic spectrum of congenital neutropenias in Israel is characterized by a high prevalence of G6PC3 variants and an absence of HAX1 mutations. Similar to other registries, for 26% of the patients, a molecular diagnosis was not achieved. However, myeloid transformation was common in this group, emphasizing the need for close follow-up.

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 severe factor XI deficiency: A multicenter study.

BACKGROUND: Factor XI (FXI) deficiency is a rare autosomal recessive bleeding disorder. Only scarce publications address its clinical features in children. The increased prevalence of FXI deficiency in Israel enabled data collection for this large multicenter cohort study. OBJECTIVE: Some hemostatic challenges may be unique or more common in children, such as bleeding in the neonatal period or trauma-related injury. The current study was designed to explore the potential impact of these differences in children with severe FXI deficiency. METHODS: Medical files of all children with FXI level under 15% followed at five tertiary centers were evaluated. The retrieved data comprised demographic and clinical characteristics, including bleeding episodes, surgical interventions, treatment strategies, as well as laboratory features. RESULTS: Sixty children, whose median age at diagnosis was 4.2 years and their median FXI level was 4%, were included. Three children experienced triggered intracranial hemorrhage (ICH) and two children had major bleeds. No bleeding complications occurred in surgeries in which hemostatic treatment consisting mostly of tranexamic acid or fresh frozen plasma was applied (n = 45). In contrast, excessive bleeding was noted in 25% of surgical procedures performed without hemostatic preparation (p = .002). CONCLUSION: This study's findings confirm the generally favorable outcome of this rare bleeding disorder, with no spontaneous bleeds or cases of perinatal ICH. Nonetheless, proper diagnosis and adequate hemostasis in the surgical setting are imperative. Unlike previous studies in adults, our pediatric study suggests an association between the severity of FXI deficiency and bleeding tendency.

Essential thrombocythemia A retrospective case series.

BACKGROUND: Essential thrombocythemia (ET) is rare in children, and pediatric guidelines are lacking. Therefore, we aimed to evaluate ET diagnosis and treatment in a pediatric cohort. PROCEDURE: Data of patients with ET from three hospitals were reviewed. Molecular diagnosis included JAK2V617F, CALR, and MPL mutations. Patients were evaluated for acquired von Willebrand syndrome (AVWS). Follow-up included clinical symptoms, adverse events, and treatment. RESULTS: Twelve children (median age: 8 years, range 1-14.5) were included. Mean lag period between the first documentation of thrombocytosis until ET diagnosis was 36 months. Six patients were positive for JAK2V617F and two for CALR mutations. In six of nine patients, AVWS was diagnosed. At diagnosis, only 33% of patients started therapy with aspirin (n = 4) and hydroxyurea (n = 2). In three of eight untreated patients, therapy was added during follow-up. The cohort was followed for a median of 32.5 months (range: 4-108 months). Clinical follow-up disclosed vascular complications in 4 of 12 patients (deep vein thrombosis, n = 1; transient ischemic attack, n = 3). Two females experienced excessive bleeding; both were diagnosed with AVWS. Neither leukemia nor myelofibrosis evolved in our cohort. CONCLUSION: Increased awareness to pediatric ET is warranted, as delayed diagnosis is common. Compared to adults, AVWS may be more prevalent among children with ET.

Characterization and genotype-phenotype correlation of patients with Fanconi anemia in a multi-ethnic population.

Fanconi anemia (FA), an inherited bone marrow failure (BMF) syndrome, caused by mutations in DNA repair genes, is characterized by congenital anomalies, aplastic anemia, high risk of malignancies and extreme sensitivity to alkylating agents. We aimed to study the clinical presentation, molecular diagnosis and genotype-phenotype correlation among patients with FA from the Israeli inherited BMF registry. Overall, 111 patients of Arab (57%) and Jewish (43%) descent were followed for a median of 15 years (range: 0.1-49); 63% were offspring of consanguineous parents. One-hundred patients (90%) had at least one congenital anomaly; over 80% of the patients developed bone marrow failure; 53% underwent hematopoietic stem-cell transplantation; 33% of the patients developed cancer; no significant association was found between hematopoietic stem-cell transplant and solid tumor development. Nearly 95% of the patients tested had confirmed mutations in the Fanconi genes FANCA (67%), FANCC (13%), FANCG (14%), FANCJ (3%) and FANCD1 (2%), including twenty novel mutations. Patients with FANCA mutations developed cancer at a significantly older age compared to patients with mutations in other Fanconi genes (mean 18.5 and 5.2 years, respectively, P=0.001); however, the overall survival did not depend on the causative gene. We hereby describe a large national cohort of patients with FA, the vast majority genetically diagnosed. Our results suggest an older age for cancer development in patients with FANCA mutations and no increased incidence of solid tumors following hematopoietic stem-cell transplant. Further studies are needed to guide individual treatment and follow-up programs.

Targeted next generation sequencing for the diagnosis of patients with rare congenital anemias.

BACKGROUND: Most patients with anemia are diagnosed through clinical phenotype and basic laboratory testing. Nonetheless, in cases of rare congenital anemias, some patients remain undiagnosed despite undergoing an exhaustive workup. Genetic testing is complicated by the large number of genes involved in rare anemias and the similarities in the clinical presentation of the different syndromes. OBJECTIVE: We aimed to enhance the diagnosis of patients with congenital anemias by using targeted next-generation sequencing. METHODS: Genetic diagnosis was performed by gene capture followed by next-generation sequencing of 76 genes known to cause anemia syndromes. RESULTS: Genetic diagnosis was achieved in 13 out of 21 patients (62%). Six patients were diagnosed with pyruvate kinase deficiency, 4 with dehydrated hereditary stomatocytosis, 2 with sideroblastic anemia, and 1 with CDA type IV. Eight novel mutations were found. In 7 patients, the genetic diagnosis differed from the pretest presumed diagnosis. The mean lag time from presentation to diagnosis was over 13 years. CONCLUSIONS: Targeted next-generation sequencing led to an accurate diagnosis in over 60% of patients with rare anemias. These patients do not need further diagnostic workup. Earlier incorporation of this method into the workup of patients with congenital anemia may improve patients' care and enable genetic counseling.

STAT3 gain-of-function mutations associated with autoimmune lymphoproliferative syndrome like disease deregulate lymphocyte apoptosis and can be targeted by BH3 mimetic compounds.

Autoimmune lymphoproliferative syndrome (ALPS) is typically caused by mutations in genes of the extrinsic FAS mediated apoptotic pathway, but for about 30% of ALPS-like patients the genetic diagnosis is lacking. We analyzed 30 children with ALPS-like disease of unknown cause and identified two dominant gain-of-function mutations of the Signal Transducer And Activator Of Transcription 3 (STAT3, p.R278H, p.M394T) leading to increased transcriptional activity. Hyperactivity of STAT3, a known repressor of FAS, was associated with decreased FAS-mediated apoptosis, mimicking ALPS caused by FAS mutations. Expression of BCL2 family proteins, further targets of STAT3 and regulators of the intrinsic apoptotic pathway, was disturbed. Cells with hyperactive STAT3 were consequently more resistant to intrinsic apoptotic stimuli and STAT3 inhibition alleviated this effect. Importantly, STAT3-mutant cells were more sensitive to death induced by the BCL2-inhibitor ABT-737 indicating a dependence on anti-apoptotic BCL2 proteins and potential novel therapeutic options.

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.

Deregulation of Fas ligand expression as a novel cause of autoimmune lymphoproliferative syndrome-like disease.

Autoimmune lymphoproliferative syndrome is frequently caused by mutations in genes involved in the Fas death receptor pathway, but for 20-30% of patients the genetic defect is unknown. We observed that treatment of healthy T cells with interleukin-12 induces upregulation of Fas ligand and Fas ligand-dependent apoptosis. Consistently, interleukin-12 could not induce apoptosis in Fas ligand-deficient T cells from patients with autoimmune lymphoproliferative syndrome. We hypothesized that defects in the interleukin-12 signaling pathway may cause a similar phenotype as that caused by mutations of the Fas ligand gene. To test this, we analyzed 20 patients with autoimmune lymphoproliferative syndrome of unknown cause by whole-exome sequencing. We identified a homozygous nonsense mutation (c.698G>A, p.R212*) in the interleukin-12/interleukin-23 receptor-component IL12RB1 in one of these patients. The mutation led to IL12RB1 protein truncation and loss of cell surface expression. Interleukin-12 and -23 signaling was completely abrogated as demonstrated by deficient STAT4 phosphorylation and interferon γ production. Interleukin-12-mediated expression of membrane-bound and soluble Fas ligand was lacking and basal expression was much lower than in healthy controls. The patient presented with the classical symptoms of autoimmune lymphoproliferative syndrome: chronic non-malignant, non-infectious lymphadenopathy, splenomegaly, hepatomegaly, elevated numbers of double-negative T cells, autoimmune cytopenias, and increased levels of vitamin B12 and interleukin-10. Sanger sequencing and whole-exome sequencing excluded the presence of germline or somatic mutations in genes known to be associated with the autoimmune lymphoproliferative syndrome. Our data suggest that deficient regulation of Fas ligand expression by regulators such as the interleukin-12 signaling pathway may be an alternative cause of autoimmune lymphoproliferative syndrome-like disease.

Genetic analysis and clinical picture of severe congenital neutropenia in Israel.

BACKGROUND: The relative frequency of mutated genes among patients with severe congenital neutropenia (SCN) may differ between various ethnic groups. To date, few population-based genetic studies have been reported. This study describes the genetic analysis of 32 Israeli patients with SCN. PROCEDURES: Clinical data were retrieved from the prospective Israeli Inherited Bone Marrow Failure Registry. Recruitment included living and deceased patients who were diagnosed between 1982 and 2012, for whom molecular diagnosis was performed. ELANE, HAX1 and G6PC3 genes were sequenced in all patients, and GFI-1 and WAS genes were sequenced if other genes were wildtype. RESULTS: Eleven patients (34%) had heterozygous mutations in ELANE (10 kindreds), eight (25%) had homozygous mutations in G6PC3 (5 kindreds) and 13 (41%) had no detected mutations. No patients had mutations in HAX1 or WAS. Four of the eight patients with G6PC3 mutations had congenital anomalies. The probability of survival for all patients was 50% at age of 18. Deaths were mainly due to sepsis (5 patients, 4/5 not responding to G-CSF, none with G6PC3 mutation). Two patients developed acute myelogenous leukemia (AML) and one myelodysplastic syndrome (MDS), none with G6PC3 mutation. CONCLUSIONS: We found a unique pattern of SCN mutations in Israel with homozygous G6PC3 mutations in eight (25%) patients, the highest frequency described so far. HAX1 mutations, reported mainly in Sweden and Iran, were absent. Patients with G6PC3 mutations had congenital anomalies, appeared to have a better response to G-CSF, and so far have not developed AML or MDS.

Preimplantation genetic diagnosis for fetal neonatal alloimmune thrombocytopenia due to antihuman platelet antigen maternal antibodies.

OBJECTIVE: To develop a reliable preimplantation genetic diagnosis protocol for antihuman platelet antigen-1 incompatibility for a family in whom antenatal treatment was not possible because of the mother's hypersensitivity to intravenous immunoglobulin (IVIG). METHODS: Haplotypes were constructed from genomic DNA of the family members. A polymerase chain reaction protocol that included eight microsatellite polymorphic markers and the ITGB3-specific (T196C, rs5918) polymorphism were multiplexed to be used in a single cell protocol, and single blastomeres were analyzed. RESULTS: In one preimplantation genetic diagnosis cycle, out of 28 retrieved oocytes, 24 embryos fertilized and 12 underwent biopsy. Three embryos were found to be antihuman platelet antigen-1b/1b homozygotes and two were transferred. This cycle resulted in an uneventful pregnancy and birth of a healthy child. CONCLUSION: In cases in which there is antihuman platelet antigen incompatibility and IVIG cannot be administered, preimplantation genetic diagnosis is a reliable alternative to enable birth of unaffected children.

Burkitt lymphoma in children: the Israeli experience.

BACKGROUND: We analyzed the results of the French-American-British-LMB 96 protocol performed in 9 centers in Israel on 88 patients with B-cell non-Hodgkin lymphoma treated from 2000 to 2005. PROCEDURE: The majority of the patients was male (63/88, 72%), with a median age of 8.9 years (range, 2.5 to 20 y). Ethnic origin was Jewish in 73% (64/88), and Arabic in 27%. Fifty (57%) patients were classified as Burkitt lymphoma, 5 (5.7%) as Burkitt-like lymphoma, 22 (25%) as diffuse large B cell (DLBC), and 9 (10.2%) as Burkitt leukemia with over 25% of their bone marrow (BM) involved. Initial disease sites included the abdomen in 43%, head and neck in 45%, and mediastinum in 7%. Stage I: 9.1%; stage II: 28.4%; stage III: 45.5%, stage IV: 17%. Two patients had BM involvement alone, 5 patients had central nervous system (CNS) involvement alone, and 4 had both CNS and BM. The children were divided into 3 groups according to risk factors, with 5 in group A, 69 in group B, and 14 in group C. RESULTS: With a median follow-up of 3 years (12 mo to 7.6 y), the Kaplan-Meier for event-free survival (EFS) and overall survival (OS) according to whole group treatment was 88.6% and 90.9%, group A was 100% and 100%; group B was 89.9% and 92.8%; and group C was 78.6% and 78.6%. There were no untoward events or deaths in group A, whereas 6 patients relapsed in group B, 4 of whom died (all relapsed during the first year), with tumor lysis syndrome in 3 patients and death of toxicity in 1 patient who had multiorgan failure 2 days after initiation of COP. Three patients in group C relapsed and died (all patients relapsed during the first 6 months), with tumor lysis syndrome in 4 patients but no deaths from toxicity. EFS for LDH less than twice was 96.4%, EFS for LDH more than twice was 73.3% (P=0.002). OS according to primary site: bone and ovary: 100%; head and neck: 95%; abdomen: 92%; mediastinum: 50%. The difference between the mediastinal primary site to all other primary sites was statistically significant with P=0.003. All the mediastinal tumors were of DLBC origin but no significant differences in outcome were found when DLBC was compared with other histologies (DLBC: 81.8%, other B line: 90.9%). OS for patients of Arabic ethnic origin was 79.2%, for Jewish patients was 95.3%, P=0.02. We could not determine any prognostic factors that were different between the groups, which raises the question of a genetic influence. CONCLUSIONS: In nonresected mature B-cell lymphoma of childhood and adolescence with no BM or CNS involvement, a 93% cure rate can be achieved, similar to the French-American-British/LMB 96 trial. Patients with primary DLBC mediastinal mass had a significantly reduced OS, indicating the need for a different therapeutic approach.

High incidence of early cholelithiasis detected by ultrasonography in children and young adults with hereditary spherocytosis.

OBJECTIVES: Retrospective cholecystography studies in adults with hereditary spherocytosis (HS) suggested detectable gallstones in 37% to 43% of patients. Since longitudinal studies using biliary ultrasonography are unavailable, the aim of the present study was to determine the incidence of gallstone disease, as detected by biliary ultrasonography, in children and young adults with HS. As individuals with HS who co-inherit Gilbert syndrome have a greater risk of developing gallstones, uridine diphosphate-glucuronyl transferase (UGT-1A) gene polymorphism was also determined. PATIENTS AND METHODS: The authors retrospectively evaluated 44 patients aged 1.4 to 22 years with HS, 12 (27%) of whom underwent splenectomy. Ultrasonography was performed annually starting at the age of 4 years or at the time of diagnosis, if later. RESULTS: Of the 44 patients, 18 (41%) developed cholelithiasis as demonstrated by gallbladder ultrasonography. In most patients (94%) the test first proved positive at age 4 to 13 years. Patients with HS and Gilbert syndrome tended to be younger at the time of cholelithiasis. CONCLUSIONS: Early cholelithiasis was detected in children and young adults with HS. To identify this complication, the authors recommend early annual biliary ultrasonography in HS children, starting at about 4 years of age. In patients with Gilbert syndrome, closer follow-up may be indicated.

Isolated extrarenal papillary carcinoma in a child.

A previously healthy 12-year-old boy presented with a mass in the right retroperitoneum adjacent to an ipsilateral hypoplastic kidney. Complete resection of the tumor and kidney were done. The tumor was found to be papillary adenocarcinoma lying 3 cm apart from a contracted kidney, which was free of tumor. No adjuvant therapy was given. After 50 months of follow-up the child is healthy without evidence of tumor recurrence.