Diamond Blackfan anemia is mediated by hyperactive Nemo-like kinase.

Diamond Blackfan Anemia (DBA) is a congenital bone marrow failure syndrome associated with ribosomal gene mutations that lead to ribosomal insufficiency. DBA is characterized by anemia, congenital anomalies, and cancer predisposition. Treatment for DBA is associated with significant morbidity. Here, we report the identification of Nemo-like kinase (NLK) as a potential target for DBA therapy. To identify new DBA targets, we screen for small molecules that increase erythroid expansion in mouse models of DBA. This screen identified a compound that inhibits NLK. Chemical and genetic inhibition of NLK increases erythroid expansion in mouse and human progenitors, including bone marrow cells from DBA patients. In DBA models and patient samples, aberrant NLK activation is initiated at the Megakaryocyte/Erythroid Progenitor (MEP) stage of differentiation and is not observed in non-erythroid hematopoietic lineages or healthy erythroblasts. We propose that NLK mediates aberrant erythropoiesis in DBA and is a potential target for therapy.

Updated analysis: central venous access device infection rates in an expanded cohort of paediatric patients with severe haemophilia receiving prophylactic recombinant tissue plasminogen activator.

INTRODUCTION: Central venous access devices (CVADs) are used in the care of paediatric haemophilic patients with difficult peripheral access, but their use is limited by complications such as infection. We previously published our experience with monthly recombinant tissue plasminogen activator (r-tPA) administration to CVADs of haemophilic patients as an intervention for infection prophylaxis, which suggested a 10-fold decrease in infection rate compared to published rates without r-tPA. AIM: This study was conducted to assess the CVAD infection rate in an expanded haemophilia cohort receiving r-tPA over an extended period. METHODS: A retrospective review was performed on patients with haemophilia who received monthly r-tPA to CVADs, with data collected from January 1, 2008 to December 31, 2012. The data were merged with the previously reported data set (collected from June 1, 1998 to December 31, 2007). RESULTS: Over the entire observation period, there were 46 350 CVAD days among 32 patients [26 severe factor VIII (FVIII) deficiency, six severe FIX deficiency]. Eight patients received immune tolerance therapy for inhibitors and 24 patients received prophylactic factor administration. No patients were HIV positive. Three infections were observed, with an overall infection rate of 0.06 infections per 1000 CVAD days. CONCLUSIONS: A low CVAD infection rate, similar to that observed in our previous study (0.04 per 1000 CVAD days), was observed in this expanded haemophilia cohort treated with prophylactic r-tPA, supporting the use of monthly r-tPA as CVAD infection prophylaxis in haemophilia patients.

Monthly recombinant tissue plasminogen activator administration to implantable central venous access devices decreases infections in children with haemophilia.

Central venous access devices (CVAD) have been effectively used in the care of haemophilia patients. This is particularly true in children, who often have difficult venous access. CVAD complications (infection and thrombosis), risk factors, and complication rates, have been well-documented. However, effective interventions which decrease complication rates have not been identified. In this study, we review our experience with the use of monthly recombinant tissue plasminogen activator (rtPA) administration in haemophilia patients with fully implanted CVADs. Data on 19 haemophilia patients with 24 CVADs were available for analysis, with a total of 24 520 CVAD days. An infection rate of 0.04 infections per 1000 CVAD days and a thrombosis rate of 0.04 thrombi per 1000 CVAD days was observed. The observed infectious complication rate is at least one logarithm lower than many published CVAD infection rates in haemophilia patients who have not received rtPA administration. No bleeding complications were noted. Monthly rtPA is safe and appears to be effective in decreasing CVAD infection rates. Larger, randomized controlled studies are indicated to validate this finding.

Bilateral microtia and cleft palate in cousins with Diamond-Blackfan anemia.

We report on maternal first cousins with bilateral microtia, micrognathia, cleft palate and hematologic findings of Diamond-Blackfan anemia (DBA). The similarity of findings shared between our cases and a female reported by Hasan and Inoue [1993] suggests that this is a distinctive syndrome, rather than a chance association. DBA is a heterogeneous disorder, caused in about 25% of cases by heterozygous mutations in the RPS19 gene (DBA1). Mutation analysis in our cases did not show an RPS19 mutation, and 2 alleles were present in each. Segregation analysis for DBA1 on chromosome 19 and DBA2 on 8p23 was not consistent with linkage. We conclude that this syndrome of microtia, cleft palate and DBA is not allelic to known DBA loci.

Evidence for linkage of familial Diamond-Blackfan anemia to chromosome 8p23.3-p22 and for non-19q non-8p disease.

Diamond-Blackfan anemia (DBA) is a rare congenital hypoplastic anemia that usually presents early in infancy and is inherited in 10% to 20% of cases. Linkage analysis has shown that DBA in many of both dominant and recessive DBA families mapped to chromosome 19q13.2 leading to the cloning of a gene on chromosome 19q13.2 that encodes a ribosomal protein, RPS19. However, subsequently, mutations of the RPS19 gene have only been identified in 25% of all patients with DBA. This study analyzed 14 multiplex DBA families, 9 of which had 19q13.2 haplotypes inconsistent with 19q linkage. A genome-wide search for linked loci suggested the presence of a second DBA locus in a 26.4-centimorgan (cM) interval on human chromosome 8p. Subsequently, 24 additional DBA families were ascertained and all 38 families were analyzed with additional polymorphic markers on chromosome 8p. In total, 18 of 38 families were consistent with linkage to chromosome 8p with a maximal LOD score with heterogeneity of 3.55 at D8S277 assuming 90% penetrance. The results indicate the existence of a second DBA gene in the 26.4-cM telomeric region of human chromosome 8p23.3-p22, most likely within an 8.1-cM interval flanked by D8S518 and D8S1825. Seven families were inconsistent with linkage to 8p or 19q and did not reveal mutations in the RPS19 gene, suggesting further genetic heterogeneity. (Blood. 2001;97:2145-2150)

Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector.

Pre-clinical studies in mice and haemophilic dogs have shown that introduction of an adeno-associated viral (AAV) vector encoding blood coagulation factor IX (FIX) into skeletal muscle results in sustained expression of F.IX at levels sufficient to correct the haemophilic phenotype. On the basis of these data and additional pre-clinical studies demonstrating an absence of vector-related toxicity, we initiated a clinical study of intramuscular injection of an AAV vector expressing human F.IX in adults with severe haemophilia B. The study has a dose-escalation design, and all patients have now been enrolled in the initial dose cohort (2 x 10(11) vg/kg). Assessment in the first three patients of safety and gene transfer and expression show no evidence of germline transmission of vector sequences or formation of inhibitory antibodies against F.IX. We found that the vector sequences are present in muscle by PCR and Southern-blot analyses of muscle biopsies and we demonstrated expression of F.IX by immunohistochemistry. We observed modest changes in clinical endpoints including circulating levels of F.IX and frequency of FIX protein infusion. The evidence of gene expression at low doses of vector suggests that dose calculations based on animal data may have overestimated the amount of vector required to achieve therapeutic levels in humans, and that the approach offers the possibility of converting severe haemophilia B to a milder form of the disease.

Hemolytic anemia in children.

This article provides an overview of hemolytic anemia in children. Main focus areas include acquired immune-mediated hemolysis, hemolytic anemia due to hereditary RBC disorders, hereditary hemolytic disorders caused by enzyme abnormalities, and hereditary hemolytic anemia due to hemoglobin abnormalities.

Home treatment of mild to moderate bleeding episodes using recombinant factor VIIa (Novoseven) in haemophiliacs with inhibitors.

OBJECTIVE: To assess the safety and efficacy of a fixed dose of recombinant activated factor VII (rFVIIa; NovoSeven) in the home setting for mild to moderately severe joint, muscle; and mucocutaneous bleeding episodes in patients with haemophilia A or B with inhibitors. DESIGN: Multicentre, open-label, single arm, phase III study of one year duration. METHODS; Patients or their caregivers administered up to three doses of rFVIIa (90 microg/kg i.v.) at 3 h intervals within 8 h of the onset of a mild to moderate bleeding episode. Once the subject considered that rFVIIa had been "effective" with regard to haemostasis (after 1-3 injections), one further (maintenance) dose of rFVIIa was administered. RESULTS: Of 60 patients enrolled, 56 experienced at least one bleed, and 46 completed the one year study. 614 of 877 bleeds (70%) were evaluable according to protocol definitions. Haemostasis was rated as "effective" in 92% (566/614) of evaluable bleeds after a mean of 2.2 injections. For successfully treated episodes, the time from onset of bleeding until administration of the first injection was 1.1+/-2.0 h (mean+/-SD). Twenty-four hours after initial successful response, haemostasis was reported as having been maintained in 95% of cases. Efficacy was comparable for muscle, joint and target joint, and mucocutaneous bleeding episodes. In an intent-to-treat analysis of all 877 bleeding events, efficacy outcomes were equivalent to the evaluable bleeds, with an effective response in 88% of treated episodes. Treatment-related adverse events occurred in 32 (3% of all) bleeding episodes and consisted of re-bleeds/new bleeds in more than 50% (18/32) of these events. A single episode of superficial thrombophlebitis was the only thrombotic complication encountered, and there were no patient withdrawals due to adverse events. Development of FVII(a) antibodies could not be detected, and hypersensitivity reactions to rFVIIa were not reported. CONCLUSION: rFVIIa is effective and well tolerated when used in the home setting to treat mild to moderate bleeding episodes in patients with haemophilia A or B with inhibitors.

Loss of elbow and wrist motion in hemophilia.

The longitudinal changes in elbow and wrist motion for 48 patients with hemophilia were reviewed to determine the effect of recurrent hemarthroses. The average age of the patients at the time of followup was 23 years 9 months. The average duration of followup was 10.8 years. The patients were divided into 3 age groups: younger than age 15 years (14 patients), age 15 to 25 years (11 patients), and older than age 25 years (23 patients). For patients older than age 25 years, pronation, supination, elbow flexion and extension, wrist flexion and extension, and ulnar deviation were significantly decreased relative to patients younger than age 15 years. Pronation was the first motion to show a significant change, decreasing by 19% in patients age 15 to 25 years and by 31% in patients older than age 25 years. Loss of elbow extension showed the greatest change. In cases of severe hemophilic arthropathy of the elbow, synovectomy and radial head excision decreased elbow pain and bleeding episodes and improved supination and pronation.

Hematologic disorders in children from southeast Asia.

The overall laboratory features of the common RBC disorders occurring in Southeast Asians is summarized in Table 4. These erythrocyte disorders will continue to be important public health issues, and it has been predicted that most new cases of thalassemia in the United States will occur in this population group. The fertility rate in Southeast Asian families is very high, with an average of more than five children delivered by each married woman. This number of children is consistent with perceptions of ideal family size, and, to date, no evidence suggests any change in the size of Southeast Asian families who now reside in the United States. Moreover, attitudes about health care, reasons why one seeks medical attention, and a variety of other cultural issues may impair the effectiveness of genetic counseling and other preventive measures designed to reduce the incidence of serious blood diseases. Genetic screening and prenatal diagnosis clearly have led to a markedly decreased incidence of homozygous thalassemia disorders in high-risk Mediterranean populations throughout the world. With further assimilation into Western culture, a similar disease may occur in the Southeast Asian population also.

Frequency of inhibitor development in haemophiliacs treated with low-purity factor VIII.

Clinical studies evaluating highly purified monoclonal-antibody-derived and recombinant-DNA-derived clotting factor concentrates in previously untreated (PUPS) severe factor VIII (FVIII) deficient haemophilia patients, have documented an increased frequency of inhibitors compared with that seen in patients who have received less pure products. However, a valid comparison of inhibitor frequency in patients treated with pure and less pure products has not been possible because appropriate studies have not been done in PUPS treated with the less pure products. To determine the frequency of inhibitor development in PUPS treated solely with less pure plasma-derived products (specific activities < 5 FVIII U/mg protein), we reviewed the records of all haemophilia patients born between 1975 and 1985 and treated with such products at any of seven centres. 89 patients with severe FVIII deficiency (< 1%) were observed and tested for inhibitors from birth to 5 years old or until 30 bleeding episodes had been treated. 25 of the 89 patients developed inhibitors (28%), and 21 of these 25 were high-titre responders (> 5 Bethesda units). This frequency of inhibitor development is greater than that reported in patients treated with monoclonal FVIII products, but the latter patients may not have been followed as long as the patients in our report. Our data may make possible a meaningful comparison with the frequency of inhibitor development in PUPS treated solely with recombinant-DNA-derived FVIII.

Langerhans' cell histiocytosis presenting with the superior vena cava syndrome: a case report.

A case of Langerhans' cell histiocytosis confined to the mediastinum and presenting with de novo superior cava syndrome is reported. The causes of superior vena cava syndrome in childhood are discussed as is the importance of obtaining pathologic diagnosis prior to initiating therapy.

Arthropathy of the ankle in hemophilia.

Seventy-five patients who had hemophilia were followed clinically and roentgenographically to assess the prevalence of hemarthrosis and the prevalence and severity of arthropathy of the ankle. The mean age of the patients at the time of follow-up was twenty-two years and seven months. The patients were divided into four age-groups: less than ten years (eleven patients), ten to nineteen years (twenty-one patients), twenty to thirty years (twenty-four patients), and more than thirty years (nineteen patients). Intra-articular bleeding occurred more frequently in the joints of the lower extremities than in the joints of the upper extremities. During the second decade of life, hemarthroses occurred more often in the ankle than in the knee. A history of recurrent bleeding into the ankle joint, chronic synovitis, and overgrowth of the medial portion of the distal tibial epiphysis was associated with an early onset of arthropathy. In older patients, compression arthrodesis of the ankle joint was helpful in eliminating pain, recurrent bleeding, and equinus deformity.

Congenital hypoplastic (Diamond-Blackfan) anemia in seven members of one kindred.

Congenital hypoplastic (Diamond-Blackfan) anemia is a rare macrocytic anemia, generally presenting during infancy or childhood. The condition usually occurs sporadically or in a pattern consistent with autosomal recessive inheritance, although autosomal dominant transmission has been proposed in some kindreds. We report the largest known kindred of congenital hypoplastic anemia, with at least 7 affected individuals over 3 generations, and propose that studies of this kindred may be useful for identifying the mechanism by which their genetic abnormality results in congenital hypoplastic anemia. Erythropoietic investigations on relatives show no inhibitors of erythropoiesis in serum, T-lymphocytes, or macrophages. Their erythroid progenitor cells (CFU-E and BFU-E) were generally quantitatively normal, and were capable of rapid proliferation, as judged by cell-cycle shortening. However, their erythroid progenitors displayed a relative insensitivity to recombinant erythropoietin, and produced relatively few normoblasts per erythroid progenitor cell. We propose that these and subsequent studies may be helpful in selecting candidate genes responsible for the molecular defect in this kindred.

SUP-HD1: a new Hodgkin's disease-derived cell line with lymphoid features produces interferon-gamma.

A new cell line, SUP-HD1, was established from the pleural effusion of a patient with nodular sclerosing Hodgkin's disease (NSHD). The SUP-HD1 cells had the characteristic morphology of Reed-Sternberg cells and contained acid phosphatase and nonspecific esterase. The cells lacked the Epstein-Barr virus (EBV) genome and reacted with monoclonal antibodies (MoAbs) against CD15 (Leu-M1), CD25 (Tac), CD71 (OKT9), Ki67, and HLA-Dr. However, the SUP-HD1 cells were nonreactive with MoAbs that specifically identify T lymphocytes, B lymphocytes, and macrophage/myeloid cells. Karyotype analysis of the cell line showed clonal abnormalities involving 1p13, 7p15, 8q22, and 11q23, chromosomal locations, at which breakpoints have been reported in HD. Southern blot analysis demonstrated rearrangement of the immunoglobulin heavy chain and kappa light chain genes as well as the gene for the beta chain of the T-cell receptor (TCR). Transcriptional analysis showed expression of RNAs for kappa light chain, interferon-gamma (IFN-gamma), and interleukin-2 receptor (IL-2R) but not IL-2. The SUP-HD1 cells lacked cytoplasmic and surface immunoglobulin heavy chain, but a small amount of cytoplasmic kappa light chain was detected. The presence of nuclear factor kappa B (NF kappa B), a B-lymphocyte-associated transcription factor, was demonstrated in stimulated and unstimulated cells. In addition, the SUP-HD1 cell line, produced IFN-gamma, a T-lymphocyte-associated lymphokine. Based on these data, the SUP-HD1 cells appear to be aberrant lymphocytes with characteristics of both activated B and T lymphocytes. Elaboration of lymphokines such as IFN-gamma by the malignant cells may represent one explanation for the unique clinical and pathologic features of HD.