Corrina McMahon

A Novel Primary Immunodeficiency with Specific Natural-Killer Cell Deficiency Maps to the Centromeric Region of Chromosome 8

We describe four children with a novel primary immunodeficiency consisting of specific natural-killer (NK) cell deficiency and susceptibility to viral diseases. One child developed an Epstein-Barr virus-driven lymphoproliferative disorder; two others developed severe respiratory illnesses of probable viral etiology. The four patients are related and belong to a large inbred kindred of Irish nomadic descent, which suggests autosomal recessive inheritance of this defect. A genomewide scan identified a single 12-Mb region on chromosome 8p11.23-q11.21 that was linked to this immunodeficiency (maximum LOD score 4.51). The mapping of the disease-causing genomic region paves the way for the identification of a novel pathway governing NK cell differentiation in humans.

Using the eosin‐5‐maleimide binding test in the differential diagnosis of hereditary spherocytosis and hereditary pyropoikilocytosis

Flow cytometric analysis of eosin‐5‐maleimide (EMA)‐labeled red blood cells (RBCs) has been used as a screening test for the diagnosis of patients with hereditary spherocytosis (HS). We assessed the fluorescence profiles for patients having HS and hereditary pyropoikilocytosis (HPP) together with their red cell indices.

Central venous access devices in children with congenital coagulation disorders: complications and long-term outcome.

Reliable venous access is essential to facilitate the administration of prophylactic factor concentrate or blood products in children with congenital coagulation disorders and immune tolerance therapy (ITT) regimens in those who develop high responding inhibitors. Poor venous access is even more problematic in very young children, the vast majority of whom will require the insertion of central venous access devices (CVADs). Previous studies have suggested that infection rates are low and that there are few long‐term complications associated with CVAD usage. We have reviewed 86 CVADs that have been inserted, since 1988, in 58 children with congenital bleeding disorders, aged 6 d to 16·5 years, attending Great Ormond Street Hospital, London, and the National Childrens Hospital, Dublin. The devices have remained in situ for 2 weeks to 92 months (median 22·5 months). Early (0–2 weeks) complications of CVAD insertion included nine bleeding episodes, one extravasation of factor concentrate, three allergic reactions to factor concentrate and five catheter infections. Overall, CVAD infection was the commonest problem encountered, with 52 devices (60%) becoming infected. Twenty‐seven CVADs (31%) required removal. Infection rates in children without inhibitors (29/68) were 1/20 patient–months or 1·6 infections/1000 patient–days, but infection rates for those with inhibitors were 1/8·5 patient–months or 4·3/1000 patient–days. Staphylococcus epidermidis was the predominant organism (25/52) isolated. Blockage of CVAD (four) and catheter disconnection (four) were the most frequently occurring non‐infectious long‐term complications. Skin erosion of the port was also seen in three children, in one child at 20 months, in one at 29 months and in one at 34 months after insertion. This study demonstrates a high CVAD infection rate and highlights the long‐term complications of CVAD usage.

Increased angiogenesis in bone marrow of children with acute lymphoblastic leukaemia has no prognostic significance

Summary. The importance of angiogenesis for the growth and viability of solid tumours has been established. Similarly, prognostic information may be gained from the extent of angiogenesis in these tumours. Haematopoietic malignancies should have equal requirements for angiogenesis and important prognostic information may be derived from quantification of bone marrow angiogenic activity. We retrospectively investigated 82 bone marrow trephine biopsies from 41 children with acute lymphoblastic leukaemia (ALL) at diagnosis and following treatment. Nine normal bone marrow trephines from age‐matched children were also analysed as controls. The microvessels were stained immunohistochemically with anti‐Factor VIII‐related antigen (antivWF) and antithrombomodulin (anti‐THR). Angiogenesis was quantified manually by two independent observers and was highly reproducible (Pearsons r = 0·91). Staining with anti‐vWF and anti‐THR was highly specific for microvessels and thetwo stains closely correlated (r = 0·68). Microvessel densities (MVD) at presentation were significantly increased in the majority of patients in comparison with controls (P < 0·0001) and MVD dropped towards normal in remission (P < 0·0001). Of interest, the difference in total vessel counts between leukaemic and normal/remission marrows was contributed solely by small microvessels. There was no significant difference in MVD at presentation or remission from children in poor prognostic groups or those who subsequently relapsed. Similarly, we could not find an association with age, sex, cytogenetic abnormality or disease phenotype.

The role of primary prophylactic factor replacement therapy in children with severe factor X deficiency

Summary. Severe factor X (FX) deficiency is one of the severest inherited coagulation disorders. Clinical manifestations include umbilical cord, mucosal, joint and central nervous system bleeding. Four Irish children with severe FX deficiency presented with umbilical cord bleeding. One developed an intraperitoneal haemorrhage and another an intracranial bleed. Prophylaxis, using intermediate purity Factor IX concentrate, was commenced within the first month of life, necessitating the insertion of central venous access devices in two of the children. All children have normal joint function, suggesting that prophylaxis commenced early in life reduces the incidence of arthropathy and improves quality of life.

South-east Asian ovalocytosis and the cryohydrocytosis form of hereditary stomatocytosis show virtually indistinguishable cation permeability defects

The hereditary stomatocytoses are a group of dominantly inherited conditions in which the osmotic stability of the red cell is compromised by abnormally high cation permeability. This report demonstrates the very marked similarities between the cryohydrocytosis form of hereditary stomatocytosis and the common tropical condition south‐east Asian ovalocytosis (SAO). We report two patients, one showing a novel cryohydrocytosis variant (Ser762Arg in SLC4A1) and a case of SAO. Both cases showed a mild haemolytic state with some stomatocytes on the blood film, abnormal intracellular sodium and potassium levels which were made markedly abnormal by storage of blood at 0°C, increased cation ‘leak’ fluxes at 37°C and increased Na+K+ pump activity. In both cases, the anion exchange function of the mutant band 3 was destroyed. Extensive electrophysiological studies comparing the cation leak and conductance in Xenopus laevis oocytes expressing the two mutant genes showed identical patterns of abnormality. These data are consistent with the cryohydrocytosis form of hereditary stomatocytosis and we conclude that the cation leak in SAO is indistinguishable from that in cryohydrocytosis, and that SAO should be considered to be an example of hereditary stomatocytosis.

Recessive congenital methaemoglobinaemia: functional characterization of the novel D239G mutation in the NADH-binding lobe of cytochrome b5 reductase

Type I recessive congenital methaemoglobinaemia (RCM), caused by the reduced form of nicotinamide adenine dinucleotide (NADH)‐cytochrome b5 reductase (cytb5r) deficiency, manifests clinically as cyanosis without neurological dysfunction. Two mutations, E255‐ and G291D, have been identified in the NADH‐binding lobe of cytb5r in previously reported patients, and we have detected a further novel mutation, D239G, in this lobe in two unrelated Irish families. Although one family belongs to the genetically isolated Traveller Community, which separated from the general Irish population during the 1845–48 famine, the D239G mutation was present on the same haplotype in both families. Three known cytb5r mutations were also identified, including the R159‐ mutation, which causes loss of the entire NADH‐binding lobe and had previously been reported in an individual with type II RCM. Characterization of the three NADH‐binding lobe mutants using a heterologous expression system revealed that all three variants retained stoichiometric levels of flavin adenine dinucleotide with spectroscopic and thermodynamic properties comparable with those of native cytb5r. In contrast to the E255‐ and G291D variants, the novel D239G mutation had no adverse impact on protein thermostability. The D239G mutation perturbed substrate binding, causing both decreased specificity for NADH and increased specificity for NADPH. Thus cytb5r deficient patients who are heterozygous for an NADH‐binding lobe mutation can exhibit the clinically less severe type I phenotype, even in association with heterozygous deletion of the NADH‐binding lobe.

Successful use of protease inhibitors in HIV‐infected haemophilia patients

The haemophilias are a group of inherited haemostatic disorders that require regular clotting factor replacement therapy in the severe and moderately severe subgroups. Prior to the introduction of adequate viral inactivation methods in 1985, haemophilia patients were at exceptionally high risk of contracting blood‐borne viruses from factor concentrates as each batch was derived from the plasma of thousands of donors. As a result, approximately 60% of these patients were infected with HIV between 1979 and 1985, and HIV infection now contributes significantly to the morbidity and mortality seen in this group.

CALR mutations are rare in childhood essential thrombocythemia

To the Editor: The incidence of childhood myeloproliferative neoplasms is significantly lower than that in adults. Also, the common JAK2 V617F mutation and other MPN-associated mutations are detected less frequently [1] inferring differences in both the etiology and molecular pathogenesis of these diseases in adults and children. Two recent reports have identified acquired insertion/deletion mutations of CALR, exclusively in exon 9, in approximately 70–80% of adult essential thrombocythemia (ET) and primary myelofibrosis (PMF) that do not possess JAK2 orMPL mutations by exome sequencing approaches. Furthermore they have demonstrated a previously unrecognized molecular complexity to these malignancies [2,3]. These two studies prompted the investigation of CALR mutations in pediatric MPN. The presence of CALR exon 9 mutations was retrospectively investigated using fluorescent PCR fragment analysis [2] in the blood or bone marrow DNA from a relatively small single-center cohort of unrelated, sporadic pediatric MPN classified according to established hematological, histo-morphological and molecular criteria [4]. This comprised of JAK2 V617F-negative ET (n1⁄4 4), JAK2 V617F-positive ET (n1⁄4 2) (Table I), JAK2 V617F-positive polycythemia vera (PV; n1⁄4 1), JAK2 V617F-negative polycythemia (n1⁄4 2), and reactive thrombocytosis (n1⁄4 8). All MPN patients had noMPLW515L or W515Kmutations. Two different mutations found in adult MPN acted as positive controls. CALR mutations were not detected in the four JAK2 V617F-negative ET patients. Also, no CALR mutations were detected in three JAK2 V617Fpositive MPN patients or in the eight children with reactive thrombocytosis (Table I). While this brief study demonstrates absence of CALR exon 9 mutations in JAK2V617F-positive childhood MPN, no CALR exon 9 mutations were found in the four JAK2 V617F-negative children with ET. If the molecular pathogenesis of pediatric and adult MPN was similar then one might expect to detect CALR mutations in a certain proportion of young JAK2and MPL-negative ET patients. Whether CALR mutations are present and will have a role in the diagnosis and classification of childhoodMPN requires verification in a larger cohort. This study serves to highlight another potential biological difference between childhood and adult MPN.

Composition and significance of splenic Gamna-Gandy bodies in sickle cell anemia

Children with sickle cell anemia may undergo acute splenic sequestration. Splenectomy is performed in an attempt to reduce further events. Histologic studies of spleens have revealed the presence of granuloma-like nodules, known as Gamna-Gandy bodies with amorphous inclusions; however, their significance is unknown. The medical case records and histologic samples of consecutive children with sickle cell anemia treated with splenectomy between 2001 and 2007 at Our Ladys Childrens Hospital, Dublin, were reviewed. Seventeen patients were identified. Gamna-Gandy bodies were studied by scanning electron microscopy and x-ray fluorescence spectroscopy. Gamna-Gandy bodies were identified in 7 (41%) patients, and amorphous inclusions were always seen. Patient age correlated significantly with Gamna-Gandy bodies (P = .002). Scanning electron microscopic analysis demonstrated the crystalline nature of Gamna-Gandy bodies and the chemical composition (C 47.1%; O(2) 29.7%; P 9.0%; K(+) 0.4%; Ca(2+) 6.4%; Fe(2+) 7.4%), whereas x-ray diffraction studied the structure (CaPO(4) ∙ FeOH). A crystal-formation gradient was observed, increasing from the red pulp to the white pulp. Our study shows that Gamna-Gandy bodies contain crystals and that their formation is age dependent. We also demonstrated the crystal structure and chemical composition and the relationship between Gamna-Gandy bodies and chest crises presplenectomy or postsplenectomy.