A Novel Biallelic LCK Variant Resulting in Profound T-Cell Immune Deficiency and Review of the Literature.

Lymphocyte-specific protein tyrosine kinase (LCK) is an SRC-family kinase critical for initiation and propagation of T-cell antigen receptor (TCR) signaling through phosphorylation of TCR-associated CD3 chains and recruited downstream molecules. Until now, only one case of profound T-cell immune deficiency with complete LCK deficiency [1] caused by a biallelic missense mutation (c.1022T>C, p.L341P) and three cases of incomplete LCK deficiency [2] caused by a biallelic splice site mutation (c.188-2A>G) have been described. Additionally, deregulated LCK expression has been associated with genetically undefined immune deficiencies and hematological malignancies. Here, we describe the second case of complete LCK deficiency in a 6-month-old girl born to consanguineous parents presenting with profound T-cell immune deficiency. Whole exome sequencing (WES) revealed a novel pathogenic biallelic missense mutation in LCK (c.1393T>C, p.C465R), which led to the absence of LCK protein expression and phosphorylation, and a consecutive decrease in proximal TCR signaling. Loss of conventional CD4+ and CD8+ αßT-cells and homeostatic T-cell expansion was accompanied by increased γδT-cell and Treg percentages. Surface CD4 and CD8 co-receptor expression was reduced in the patient T-cells, while the heterozygous mother had impaired CD4 and CD8 surface expression to a lesser extent. We conclude that complete LCK deficiency is characterized by profound T-cell immune deficiency, reduced CD4 and CD8 surface expression, and a characteristic TCR signaling disorder. CD4 and CD8 surface expression may be of value for early detection of mono- and/or biallelic LCK deficiency.

Immunodeficiency associated with a novel functionally defective variant of SLC19A1 benefits from folinic acid treatment.

Insufficient dietary folate intake, hereditary malabsorption, or defects in folate transport may lead to combined immunodeficiency (CID). Although loss of function mutations in the major intestinal folate transporter PCFT/SLC46A1 was shown to be associated with CID, the evidence for pathogenic variants of RFC/SLC19A1 resulting in immunodeficiency was lacking. We report two cousins carrying a homozygous pathogenic variant c.1042 G > A, resulting in p.G348R substitution who showed symptoms of immunodeficiency associated with defects of folate transport. SLC19A1 expression by peripheral blood mononuclear cells (PBMC) was quantified by real-time qPCR and immunostaining. T cell proliferation, methotrexate resistance, NK cell cytotoxicity, Treg cells and cytokine production by T cells were examined by flow cytometric assays. Patients were treated with and benefited from folinic acid. Studies revealed normal NK cell cytotoxicity, Treg cell counts, and naive-memory T cell percentages. Although SLC19A1 mRNA and protein expression were unaltered, remarkably, mitogen induced-T cell proliferation was significantly reduced at suboptimal folic acid and supraoptimal folinic acid concentrations. In addition, patients' PBMCs were resistant to methotrexate-induced apoptosis supporting a functionally defective SLC19A1. This study presents the second pathogenic SLC19A1 variant in the literature, providing the first experimental evidence that functionally defective variants of SLC19A1 may present with symptoms of immunodeficiency.

Neonatal Lymphopenia Screening Is Important for Early Diagnosis of Severe Combined Immunodeficiency.

OBJECTIVE: T-cell receptor excision circles are expensive for neonatal severe combined immunodeficiency screening in developing countries. We aimed to detect immunodeficiencies presenting with lymphopenia to enable screening in the general population and to improve awareness regarding lymphopenia among clinicians. STUDY DESIGN: This study was conducted prospectively. In all newborns included, complete blood count from umbilical cord blood samples was recorded. Absolute lymphopenia was defined as absolute lymphocyte count <3,000/mm3 in umbilical cord blood sample. Complete blood count was repeated at month 1 in cases found to have lymphopenia. RESULTS: Overall, 2,000 newborns were included in the study. Absolute lymphopenia was detected in 42 newborns (2.1%), while lymphocyte count was >3,000/mm3 in 1,958 newborns (97.9%). Two infants with persisted lymphopenia at the end of the first month; therefore, further evaluations such as lymphocyte subsets for severe combined immunodeficiency (SCID) were done. In the first infant, the lymphocyte subgroups were detected as compatible with T (-), B (-), natural killer cells (NK) (+) SCID phenotype RAG defect. Sanger sequencing revealed that NM_000448 c.2209C > T (p.R737C) homozygous mutation of RAG1 gene. In the other infant, the lymphocyte subgroups were found as considered with T (-), B (+) NK (-) SCID phenotype JAK3 defect. Both patients underwent hematopoietic stem cell transplantation from human leukocyte antigen-matched family member. CONCLUSION: Absolute lymphopenia by complete blood count is a more simpler, relatively noninvasive and inexpensive screening methodfor detection of SCID in newborns compared with T-cell receptor excision circles technique. KEY POINTS: · Our study was conducted with a much smaller number of study groups compared with the previous ones.. · However, SCID was found at a higher rate compared with other studies.. · Our study for this disease that is common in our country where consanguineous marriages are common.

Evaluation of primary care physicians' approaches to hemophilia and bleeding disorders: a questionnaire survey.

Bleeding disorders are causes of great concern and panic for parents and primary care providers. Lack of knowledge and awareness on appropriate screening tests and factor product preparation contributed to potential diagnostic delays, increased complications, and economic costs. This study aimed to determine and compare the approach of primary care physicians (including general practitioners) and emergency physicians with a questionnaire including simulation-based cases on hemophilia. This simulation and two-stage questionnaire study was conducted with 244 participants. Before-after questionnaires, two case simulations, a brief presentation, and statistical analysis were performed. Participants mostly preferred tests, such as prothrombin time (PT) or partial thromboplastin time (PTT) to bleeding time for primary hemostasis (PT/PTT n : 192, 84.2%, bleeding time n : 94, 41.2%). Similar results were found for secondary hemostasis (bleeding time n : 144, 63.4%). There was a lack of knowledge in the management of simulation-based cases of acute hemorrhagic complications and factor product preparation (complication case: correct n : 100, 55.2%; initial doses correct n : 56, 43.4%, factor preparing correct n : 37, 49.3%, factor admission correct n : 36, 24.3%). All changed significantly, after the presentation ( P  = 0.000). Our study shows that there is probably a lack of knowledge of diagnostic investigations and appropriate factor product preparation with possible consequences for patients and economics.

Serum levels of soluble HLA-G correlate with disease activity in pediatric patients with Crohn's disease.

Background: Human leukocyte antigen (HLA)-G, a member of the HLA family, is crucial for fetomaternal tolerance. Transmembrane or circulating/soluble HLA-G (sHLA-G) is elevated in autoimmune conditions and the tumor microenvironment. Circulating sHLA-G levels and their association with disease activity have not yet been assessed in pediatric patients with inflammatory bowel disease (IBD). This study aimed to quantify the serum sHLA-G levels of pediatric patients with IBD and assess the association of serum sHLA-G with disease activity. Methods: : We enrolled 24 pediatric IBD patients Crohn's disease (CD) and ulcerative colitis (UC), n = 12 each] and 24 healthy controls. Based on the disease activity index, five and seven of the CD patients had mild and moderate/severe disease, respectively, whereas six of the UC patients were in remission and six had mild disease. Serum was collected and sHLA-G levels were determined by enzyme-linked immunosorbent assay (ELISA). Results: : Pediatric patients with CD had significantly higher sHLA-G levels compared with patients with UC and healthy controls. Notably, serum sHLA-G levels were significantly higher in patients with moderate/severe CD than in those with mild CD. Conclusions: : Serum sHLA-G levels correlate with disease activity in pediatric patients with CD and are higher in CD patients than in UC patients. Thus, sHLA-G is a potential biomarker for disease activity in CD.

Social exclusion and behavior problems in adolescents with cancer and healthy counterparts.

PURPOSE: This cross-sectional study was conducted to determine social exclusion, internalized and externalized behavioral problems in adolescents with cancer and to compare them with healthy counterparts. DESIGN AND METHODS: The sample consisted of adolescents age 10-19 years (N = 70) followed up in the hemato-oncology outpatient clinic of a tertiary hospital and healthy adolescents age 10-19 years (N = 92) who were studying in secondary and high schools. The data were collected with a questionnaire for adolescents with cancer and healthy adolescents, The Ostracism Experience Scale for Adolescents (OES-A), Youth Externalizing Behavior Screener (YEBS), and Youth Internalizing Problems Screener (YIPS). RESULTS: The OES-A mean scores of cancer and healthy adolescents in the study were 35.68 ± 9.38 and 27.64 ± 5.35 (p ≤ 0.001), the YEBS mean scores were 23.51 ± 4.88 and 20.52 ± 5.42 (p ≤ 0.001), and the YIPS mean scores were 21.72 ± 6.48 and 19.18 ± 7.60 (p = 0.007), respectively. There was a low-level positive correlation between the mean scores of the OES-A and YEBS (r = 0.345, p < 0.05) and mean scores of the YEBS and YIPS (r = 0.308, p < 0.05) of adolescents with cancer. CONCLUSIONS: Adolescents with cancer had higher scores on social exclusion, internalized and externalized behavioral problems than healthy counterparts in the current study. PRACTICE IMPLICATIONS: The current study should lead pediatric oncology nurses to be more aware of social exclusion and internalized and externalized behavioral problems in adolescents with cancer after clinical treatment, and to provide appropriate psycho-oncological care.

Therapeutic effects of vitamin D and IL-22 on methotrexate-induced mucositis in mice.

Mucositis is a common side effect of cancer therapies and transplant conditioning regimens. Management of mucositis involves multiple approaches from oral hygiene, anti-inflammatory, anti-apoptotic, cytoprotective, and antioxidant agents, to cryo-therapy, physical therapy, and growth factors. There is room for novel, affordable treatment options, or improvement of currently available therapies. Vitamin D has been shown to regulate mucosa-resident cell populations such as Th17 or innate lymphoid cells and critical mucosal cytokine IL-22; however, their therapeutic potential has not been put to test in preclinical mouse models. In this study, we aimed to test the therapeutic potential of vitamin D injections and IL-22 overexpression in a murine model of chemotherapy-induced mucositis. Balb/c mice were given daily intraperitoneal injections of vitamin D. Mucositis was induced by methotrexate. Another group received IL-22 plasmid via hydrodynamic gene delivery. Weight loss and intestinal histopathology, intestinal levels of cytokines IL-22, IL-17A, GM-CSF, IL-23, IFN-γ, TNF-α, and IL-10, and number of intestinal lamina propria B cell, neutrophil, and total innate lymphoid cells were quantified. Daily vitamin D injections ameliorated intestinal inflammation and elevated intestinal IL-22 levels compared with control groups. Temporal overexpression of IL-22 by hydrodynamic gene delivery slightly increased intestinal IL-22 but failed to confer significant protection from mucositis. To our knowledge, this is the first experimental demonstration in an animal model of mucositis of therapeutic use of vitamin D and IL-22 supplementation and our results with vitamin D suggest it may have merit in further trials in human mucositis patients.

A Novel Intronic Mutation Reduces HAX1 Level and is Associated With Severe Congenital Neutropenia.

Severe congenital neutropenia (SCN) is a rare disease. Autosomal recessive forms of SCN are more frequent in countries where consanguineous marriages are common. In this report, we describe a 54-day-old female with neutropenia who presented with ecthyma gangrenosum. Clinical exome sequencing was used to identify the mutation. HAX1 messenger RNA and isoforms were examined by real-time quantitative and conventional polymerase chain reaction. Bone marrow aspiration was stained by hematoxylin and eosin. Granulocytes were tested for apoptosis upon H2O2 exposure. T-cell proliferation was tested by flow cytometry. Clinical exome sequencing revealed a novel homozygous acceptor splice site mutation in intron 3 of HAX1 (c.505-1G>C), which reduced both isoforms A and B of HAX1 messenger RNA. The Western blot studies showed a complete absence of HAX1 protein. The purified neutrophils from the patient showed increased apoptosis upon H2O2 exposure, whereas T-cell proliferative responses to various stimuli were intact. The patient was treated with combined antibiotics, filgrastim, and placed on antibiotics prophylaxis. To the best of our knowledge, our data provide the first experimental evidence for HAX1 deficiency because of a splice site mutation. Although 3 other splice site variants have been deposited in databases, functional studies were missing. This novel variant of HAX1 may explain the SCN and secondary infections in our patients.

Genetic Analysis of a Cohort of 275 Patients with Hyper-IgE Syndromes and/or Chronic Mucocutaneous Candidiasis.

Hyper-IgE syndromes and chronic mucocutaneous candidiasis constitute rare primary immunodeficiency syndromes with an overlapping clinical phenotype. In recent years, a growing number of underlying genetic defects have been identified. To characterize the underlying genetic defects in a large international cohort of 275 patients, of whom 211 had been clinically diagnosed with hyper-IgE syndrome and 64 with chronic mucocutaneous candidiasis, targeted panel sequencing was performed, relying on Agilent HaloPlex and Illumina MiSeq technologies. The targeted panel sequencing approach allowed us to identify 87 (32 novel and 55 previously described) mutations in 78 patients, which generated a diagnostic success rate of 28.4%. Specifically, mutations in DOCK8 (26 patients), STAT3 (21), STAT1 (15), CARD9 (6), AIRE (3), IL17RA (2), SPINK5 (3), ZNF341 (2), CARMIL2/RLTPR (1), IL12RB1 (1), and WAS (1) have been detected. The most common clinical findings in this cohort were elevated IgE (81.5%), eczema (71.7%), and eosinophilia (62.9%). Regarding infections, 54.7% of patients had a history of radiologically proven pneumonia, and 28.3% have had other serious infections. History of fungal infection was noted in 53% of cases and skin abscesses in 52.9%. Skeletal or dental abnormalities were observed in 46.2% of patients with a characteristic face being the most commonly reported feature (23.1%), followed by retained primary teeth in 18.9% of patients. Targeted panel sequencing provides a cost-effective first-line genetic screening method which allows for the identification of mutations also in patients with atypical clinical presentations and should be routinely implemented in referral centers.

Long-Term Follow-Up of Patients with a Diagnosis of Posterior Reversible Encephalopathy Syndrome.

OBJECTIVE: The essential characteristics of posterior reversible encephalopathy syndrome (PRES) are the presence of acute onset neurologic symptoms, focal vasogenic edema at neuroimaging, and reversible clinical and/or radiologic findings. This study aimed to evaluate the clinical findings, causes, radiologic findings, and prognoses of patients with PRES. METHODS: Patients with PRES confirmed with clinical and radiologic findings by a pediatric neurologist were evaluated retrospectively. RESULTS: Seventeen patients with PRES were evaluated (mean age at onset, 10.23 ± 4.65 years; range, 2-17 years; girls, 29.4% [n = 5]). The mean length of follow-up was 6 ± 2.3 years (range, 3.4-10 years). Mortality due to primary disease occurred in 4 patients (23.5%) during follow-up. PRES was derived from renal diseases in 10 patients (58.8%), hematologic diseases in 6 patients (35.3%), and liver disease in one patient (5.9%). Hypertension was present in 16 patients (94.1%) at onset of PRES (>99th percentile). Seizure, the most frequent initial symptom, was observed in 82.4% (n = 14). Blurred vision and headache were the initial symptoms in 3 patients (17.6%). Sequelae were observed at magnetic resonance imaging (MRI) in 6 patients. Development of epilepsy was determined as a sequela in 4 patients (23.5%) and mental motor retardation in 2 patients (11.8%). CONCLUSION: Epilepsy is uncommon in patients who have recovered from PRES. The presence of gliosis on MRI and interictal epileptic discharges on electroencephalograms are major risk factors for the development of epilepsy. Antiepileptic treatment can be stopped in the early period in patients with normal MRI and electroencephalogram by eliminating the factors that trigger the seizures.

Refractory and Fatal Presentation of Severe Autoimmune Hemolytic Anemia in a Child With the DNASE1L3 Mutation Complicated With an Additional DOCK8 Variant.

Various autoimmune diseases may be associated with primary immune deficiencies. We reported a case with a loss-of-function mutation in DNASE1L3, a gene described previously in families with systemic lupus erythematosus. In addition, the patient showed a novel homozygous missense variant in DOCK8, a gene known to be responsible for the hyper-IgE recurrent infection syndrome (HIES). A 3-year-old girl born to consanguine parents presented with chronic urticarial rash, hemolytic anemia, pulmonary hemorrhage, and hypovolemic shock findings. She had a low hemoglobin level, a positive direct antiglobulin test, antinuclear antibody and anti-double stranded DNA, low C3 and C4, third-degree tricuspid regurgitation, and severe enlargement of the right ventricle on echocardiography, suggesting pulmonary embolism. Despite treatment with intravenous immunoglobulin, pulse metilprednisolone, rituximab, and supportive treatment for shock, the patient died on the seventh day. Whole-exome sequencing indicated a homozygous stop variant c.537G>A (p. Trp179Ter) in DNASE1L3. In addition, a possibly pathogenic homozygous missense variant in the HIES gene DOCK8 was detected. The occurrence of potentially clinically relevant, genetic variants in several genes posed various challenges with respect to diagnosis, treatment, and prognosis.

Type 1 Plasminogen Deficiency With Pulmonary Involvement: Novel Treatment and Novel Mutation.

Type 1 plasminogen deficiency is a rare genetic disorder. Type 1 plasminogen deficiency is characterized by fibrin-rich pseudomembrane formation on mucosal surfaces, particularly the conjunctiva. Tracheobronchial tree involvement is a less common reported manifestation of type 1 plasminogen deficiency. Pseudomembranes in the tracheobronchial tree may result in respiratory compromise and ultimately fail if not recognized and treated. Currently, there is no specific replacement therapy approved for the treatment of congenital plasminogen deficiency. In the present paper, we report that type 1 plasminogen deficiency with novel frameshift mutation and pulmonary involvement was treated initially with systemic fresh frozen plasma followed by pulmonary lavage with fresh frozen plasma and tissue plasminogen activator.

Common Variable Immunodeficiency, Autoimmune Hemolytic Anemia, and Pancytopenia Associated With a Defect in IKAROS.

OBJECTIVE: Mutations in IKZF1, which encodes Ikaros family zinc finger 1 (IKAROS) transcription factor, are associated with recurrent infections, cytopenia, autoimmune diseases, and hematologic malignancies. Diverse clinical phenotypes resulting from IKZF1 mutations include pulmonary fungal infections, cytopenia, autoimmune hemolytic anemia (AIHA), and malignancies. In this study, we aimed to assess the DNA-binding ability and pericentromeric (PC) localization of a variant of IKZF discovered in a patient. MATERIALS AND METHODS: DNA-binding ability of a pathogenic IKZF variant was tested using electrophoretic mobility shift assay and PC localization of the variant was assessed by immunofluorescent microscopy in NIH3T3 cells. RESULTS: Clinical features of a 3-month-old male infant who underwent hematopoietic stem cell transplantation because of an IKZF1 mutation-associated common variable immunodeficiency, AIHA, and pancytopenia are described. DNA studies revealed a heterozygous missense variant (IKZF1 NM_006060 c.427C>T; p.R143W). Cotransfection studies revealed that mutant R143W has a partial dominant-negative effect over PC targeting and DNA binding. CONCLUSIONS: IKZF1 mutation must be kept in mind if neonatal AIHA, common variable immunodeficiency, and pancytopenia are observed.

Vena cava superior syndrome in the children with mediastinal tumors: Single-center experience.

OBJECTIVE: Vena cava superior syndrome comprises various symptoms of compression of vena cava superior. The results of increased venous pressure in the upper body may cause edema of the head and neck associated with cyanosis, plethora and distended subcutaneous vessels. Vena cava superior syndrome is rare in childhood. Therefore, we planned this retrospective study. METHODS: The retrospective study was carried out on the children with mediastinal tumors in the Department of Pediatric Hematology-oncology, Erciyes University Faculty of Medicine, Kayseri, Turkey , from January 2010 to December 2017. Diagnostic procedures included hematological investigations, chestradiography, thoracic computed tomography, echocardiography and lymph node or mediastinal biopsy. RESULTS: In this study, 19 (five were female) of 41 patients with mediastinal tumors had Vena cava superior syndrome. Diagnosis included Hodgkin's lymphoma in seven (37%), non-Hodgkin's lymphoma in six (32%), acute T- lymphoblastic leukemia in four (21%), neuroblastoma and anaplastic round cell sarcoma in one each respectively. All of the 19 patients' facial swelling, venous distention and mediastinal widening. All patients received intravenous corticosteroids (0.6 mg/kg dexamethasone). Furthermore, the patient with anaplastic round cell sarcoma received emergency radiotherapy. No patients died because of Vena cava superior syndrome. CONCLUSION: Vena cava superior syndrome is a medical emergency that requiresurgent treatment. Vena cava superior syndrome studies in children are rare. In this retrospective study, we found that the most common cause of Vena cava superior syndrome was Hodgkin's lymphoma different from literature.

Congenital afibrinogenemia in a 4-year-old girl complicated with acute lymphoblastic leukemia.

BACKGROUND: Congenital fibrinogen deficiency is one of the rare inherited coagulation disorders. Congenital fibrinogen deficiency complicated with a hematological malignancy can be life threatening. CASE: We present a four-year-old girl with congenital fibrinogen deficiency complicated with acute lymphoblastic leukemia. CONCLUSION: This case aims to highlight therapeutic approaches for the management of afibrinogenemia patients with acute leukemia.

Author Correction: Human FCHO1 deficiency reveals role for clathrin-mediated endocytosis in development and function of T cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Role of a second transplantation for children with acute leukemia following posttransplantation relapse: a study by the Turkish Bone Marrow Transplantation Study Group.

We examined outcomes of 51 pediatric patients with relapsed acute leukemia (AL) who underwent a second allogeneic hematopoietic stem cell transplantation (alloHSCT). After a median follow-up of 941 days (range, 69-2842 days), leukemia-free survival (LFS) and overall survival (OS) at 3 years were 26.6% and 25.6%, respectively. The nonrelapse mortality rate (NMR) and cumulative incidence of relapse (CIR) were 36.4% and 42.4%, respectively. The Cox regression analysis demonstrated that the risk factors at second transplantation for predicting limited LFS were active disease (hazard ratio (HR) = 5.1), reduced intensity conditioning (RIC) (HR = 5.0), matched unrelated donor (MUD) (HR = 3.4) and performance score <80 (HR = 3.2). Pediatric patients with AL who relapsed after their first alloHSCT may survive with a second alloHSCT. Disease status, conditioning intensity, donor type, and performance score at the second transplantation are the relevant risk factors. A score based on these factors may predict the results of the second transplantation.

Human FCHO1 deficiency reveals role for clathrin-mediated endocytosis in development and function of T cells.

Clathrin-mediated endocytosis (CME) is critical for internalisation of molecules across cell membranes. The FCH domain only 1 (FCHO1) protein is key molecule involved in the early stages of CME formation. The consequences of mutations in FCHO1 in humans were unknown. We identify ten unrelated patients with variable T and B cell lymphopenia, who are homozygous for six distinct mutations in FCHO1. We demonstrate that these mutations either lead to mislocalisation of the protein or prevent its interaction with binding partners. Live-cell imaging of cells expressing mutant variants of FCHO1 provide evidence of impaired formation of clathrin coated pits (CCP). Patient T cells are unresponsive to T cell receptor (TCR) triggering. Internalisation of the TCR receptor is severely perturbed in FCHO1-deficient Jurkat T cells but can be rescued by expression of wild-type FCHO1. Thus, we discovered a previously unrecognised critical role of FCHO1 and CME during T-cell development and function in humans.