Michele D'Avanzo

Mutational screening of thrombopoietin receptor gene (c-mpl) in patients with congenital thrombocytopenia and absent radii (TAR)

Thrombocytopenia with absent radii (TAR) is a rare autosomal recessive disease characterized by hypomegakaryocytic thrombocytopenia and bilateral radial aplasia. We performed mutational screening of coding and promoter regions of the c‐mpl gene, encoding thrombopoietin (TPO) receptor, by sequence analysis in four unrelated patients affected by TAR syndrome. Our results indicate that c‐mpl gene mutations are not a common cause of thrombocytopenia in TAR syndrome.

Concealed administration of frusemide simulating Bartter syndrome in a 4.5-year-old boy

A 4.5-year-old boy was admitted to three different hospitals because of a tendency towards dehydration and polyuria, along with normal blood pressure, hypochloraemia, hypokalaemia, metabolic alkalosis and an impaired urinary concentrating ability. A renal biopsy failed to reveal juxtaglomerular hyperplasia. The clinical and laboratory findings failed to improve despite supplementation with potassium chloride and treatment with indomethacin. The urine was found to contain frusemide. The parents denied any drug administration to the boy. The child is now doing well more than 1 year after separation from his mother. Since ingestion of diuretic cannot be differentiated from true Bartter syndrome by blood and urinary electrolyte measurements alone, a diuretic screen is warranted in children with findings consistent with Bartter syndrome.

Macroamylasemia in a 5-year-old Girl

In macroamylasemia, a macromolecular complex consisting of amylase linked to immunoglobulins circulates in the plasma and usually causes benign hyperamylasemia with low or normal amylasuria. Macroamylasemia is extremely rare in pediatric patients as it has been described in only four patients. We report herein the case of a 5-year-old girl with abdominal pain and macroamylasemia. To recognize macroamylase, we used agar gel electrophoresis, PEG precipitation, and fast protein liquid chromatography (FPLC). In our case, FPLC was found to be the most reliable method for the identification of the macromolecular complex. Macroamylasemia is merely a biochemical abnormality that is not associated with any kind of pathology. Its identification is therefore essential in order to avoid a wrong diagnosis, i.e., pancreatitis, with consequent inappropriate therapies.

Spontaneous remission from RAEB in a child.

1 Flasshove M, Strumberg D, Ayscue L, Mitchell BS, Tirier C, Heit W, Seeber S, Schutte J. Structural analysis of the deoxycytidine kinase gene in patients with acute myeloid leukemia and resistance to cytosine arabinoside. Leukemia 1994; 8: 780–785. 2 Owens JK, Shewach DS, Ullman B, Mitchell BS. Resistance to 1beta-D-arabinofuranosylcytosine in human T-lymphoblasts mediated by mutations within the deoxycytidine kinase gene. Cancer Res 1992; 52: 2389–2393.

Effect of measles-mumps-rubella vaccination on polymorphonuclear neutrophil functions in children

Adherence, metabolic burst and chemotaxis of polymorphonuclear neutrophils (PMNs) were examined in 15 children before and seven days after measles‐mumps‐rubella vaccine administration. In all children, PMN functions were significantly reduced on the seventh day. Adherence, metabolic burst and chemotaxis tested in three subjects one month after vaccination had returned to normal values. Only two children presented transient hyperpyrexia. We conclude that measles‐mumps‐rubella vaccine administration suppresses PMN functions without clinical consequences. This is probably because attenuated strains of vaccine viruses do not replicate in lymphoid tissues as extensively as do wild‐type strains.

Aase-Smith syndrome: report of a new case.

The association of congenital hypoplastic anaemia and triphalangeal thumbs was first described in two male siblings by Aase & Smith (1969). To our knowledge, only eight patients (five males and three females) have been reported in the literature (Pfeiffer & Ambs, 1983). Here we describe an additional case. The patient was a female infant who achieved a prolonged clinical remission upon corticosteroid treatment. A female infant was delivered at full term. Her weight was 2.8 kg and length 49 cm. Physical examination at 3 months revealed growth retardation (weight 3.35 kg: length 53 cm), cleft soft palate, micrognatia and triphalangeal right thumb. There was no family history of haematological disease or hand deformity. The haemoglobin concentration was 8.2 g/ dl, the red cell count 2.4 x 10l2/1, reticulocytes 3%. MCV 91 fl. The white cell count was 1 2 . 4 ~ 10y/l with 38.7% neutrophils, 48.4% lymphocytes, 7.3% monocytes, 2.4% eosinophils and 1.1% basophils. The platelet count was 1020 x 1 Oy/l. The serum iron concentration was 14.7 pmol/ 1. Fetal haemoglobin concentration was determined on one occasion and was in the normal range. Chromosome studies on cultured lymphocytes showed a normal 46 XX karyotype. Dermatoglyphic analysis showed distal displacement of palmar triradii. The severe anaemic state prompted a transfusion with packed red cells. Subsequent examination revealed a slow pattern of growth that persisted until the age of 8 months (weight 3.48 kg; length 56 cm). No evidence for intestinal malabsorption was obtained. Subsequently the physical condition of the patient improved. At the age of 18 months s8he had reached 10.2 kg and her height was 72 cm. Throughout this period haemoglobin values consistently ranged between 8.5 and 9.5 g/dl. Sixteen months later, the patient was weak, looked very pale and her haemoglobin was 3.3 g/dl. The red cell count was 0.97 x 10IL/l, haematocrit 10%. retiiculocytes 3%. the WBC count 6.3 x 10y/l, platelets 430 x 1OY/1. Direct and indirect Coombs tests were negative. Glucose-~5-phosphate-dehydrogenase activity was in the normal range. No pathologic haemoglobins were found. The bone marrow aspirate showed hypoplasia of the erythroid series with normal numbers of immature cells of the other lineages and absence of infiltrating mononuclear cells. Throughout the clinical history of the patient MCV varied between 87 and 97 fl the latter value being when anaemia had reached its nadir (Hb 3.3 g/dl). This trend to macrocytosis may indicate the occurrence of dyserythropoiesis. Neutropenia was. however, never observed. The patient was transfused with packed red cells and the haemoglobin rose to 7.6 g/dl. She was subsequently treated with the corticosteroid Deflazacort (2 mg/kg/d) for 1 month. A prompt reticulocytosis (6%) was observed and the haemoglobin value increased to 11 g/dl. Anaemia recurred when the drug was discontinued but responded readily to a second course of Deflazacort (2 g/kg/d). Two months later Deflazacort was tapered to 1 mg/kg/d. Treatment with this schedule has been continued. The patient is now 4 years old and haemoglobin levels consistently range from 10 to 11 g/dl. Macrocytosis has almost completely disappeared. Mononuclear cells (MNC) were isolated from heparinized bone marrow samples by centrifugation on Fycoll-Hypaque density gradients. 1 x lo5 MNC were suspended in 1 ml of a mixture containing 0.8% methylcellulose (Dow Chemical Company, Midland, Michigan), alpha-medium (Gibco, Grand Island Biological Company, New York), 30% heat-inactivated fetal calf serum (Gibco), 10 -4 M alpha-thyglycerol, 1 % penicillin-streptomycin, and 2 IU erythropoietin (Connaught Labs, Ontario, Canada). Plates were incubated at 3 7OC in 5% C02 and scored for colony forming unit erythroid (CFU-E) after 7 d and for burst forming unit erythroid (BFU-E) after 14 d. Only benzidine-positive colonies comprised at least of eight cells for CFU-E and 50 cells for BFU-E were enumerated (Pistoia et al, 1985). 1 x lo5 bone marrow MNC were resuspended in 1 ml of a mixture containing 0.3% agar, alpha-medium, 10% heatinactivated FCS, non-essential aminoacids, L-glutamine, sodium pyruvate, and the conditioned medium of the GCT cell line (Gibco), at the final concentration of 10% (Pike & Robinson, 1980). In some experiments, the source of colony stimulating activity (CSA) was the conditioned medium of T lymphoma cell line MO (Golde et al, 1978), used at 37°C in 5% COz and scored for colonies after 14 d. A colony was defined as an aggregate containing more than 50 cells. All the above cultures were set up in triplicate: results are means fSE. Culture of bone marrow MNC from the untreated patient gave rise to 223f30x lo5 cells BFU-E 1 3 0 f 10 x lo5 cells and 2 19 f 35 x lo5 cells colony forming unit granulocyte-macrophage (CFU-GM). The numbers of CFU-GM colonies obtained with the GCT and the MO cell line conditioned media were superimposable. The above values fell into our normal range, determined by culturing control marrows from haematologically normal individuals aged between 3 months and 35 years: BFU-E 100-275 x lo5 cells: CFU-E 50-260 x lo5 cells; 14 d CFU-GM 70-230 x lo5 cells. The coexistence of congenital hypoplastic anaemia with triphalangeal thumb in the patient herein reported suggested the diagnosis of Aase-Smith syndrome. The absence of chromosomal fragility, leucopenia, and thrombocytopenia helped to rule out Fanconis syndrome. Likewise, the presence of a triphalangeal thumb in our case was monolateral similarly to that reported by Pfeiffer & Ambs (1983). The

Heterogeneity of the erythropoietic defect in two cases of aase-smith syndrome

Here we report two children with Aase-Smith syndrome (triphalangeal thumbs and congenital red cell plasia). In vitro growth of erythroid colonies was normal in the first patient and totally absent in the other. In both patients, treatment with glucocorticoids induced remission of anemia. Our results suggest that the different growth patterns of erythroid colonies observed in the two patients could reflect the defect of erythroid differentiation occurring at discrete maturational levels.

Factor VIII response to vasopressin in nephrogenic diabetes insipidus

gested that the factor VIII response to DDAVP is a useful test for diagnosing NDI and for identifying carriers. Subsequently, Ohzeki et al., 2 however, showed strong FVIIIC and FVIIIR:Ag responses after DDAVP stimulus of a neonate with NDI. We studied the increase in the levels of FVIIIR:Ag and FVIIIC after DDAVP stimulation in two NDI patients, respectively aged 2 and 5 years, and their obligate carrier mothers. DDAVP (0.30 #g/kg; maximum dose, 24 ~g) was diluted to a final concentration of 0.5 ~g/ml in physiologic saline solution and infused intravenously for 20 minutes. Venous blood samples were collected just before and 1 hour after infusion. FVIIIR:Ag was assayed by quantitative immunoeleetrophoresis with commercial monospecific antiserum (Clottimmune, aggregated human 3,-globulin-associated protein, Behringwerke, Marburg, Germany). FVIIIC was assayed in fresh samples by a one-stage method based on the partial thromboplastin time, with factor VIII-deficient plasma (Instrumentation Laboratories, Lexington, Ky.) used as substrate. Factor VIII responses were compared with mean _ 2 SD for the control group (z score). As shown in the Table, after DDAVP stimulation the first patient had no marked increase in either FVIIIC or FVIIIR:Ag levels; the second patient had a normal increase in the FVIIIC level but no FVII1R:Ag response. In the two obligate carriers, as in the second patient, there was a normal increase of FVIIIC after stimulus but no increase of FVIIIR:Ag. Our results partially differ from those of Kobrinsky. A high response of FVIIIR:Ag and FVIIIC to DDAVP stimulus was also found in the patient reported by Ohzeki. In the obligate carriers whom we studied, the FVIIIC response to DDAVP did not significantly differ from that of control subjects, but the FVIIIR:Ag response was significantly reduced in the first carrier (10% of reference values) and was completely absent in the second. On the basis of these results, one can assume NDI to be a heterogeneous illness, at least regarding the FVIII response to DDDAVP stimulus. Caution should therefore be exercised in using the test for identifying carriers or diagnosing the illness.

Decreased adherence of polymorphonuclear neutrophils in children with viral infection.

ABSTRACT. The adherence of polymorphonuclear neutrophils was examined in 16 children affected by enteritis, pneumonia, hepatitis and infectious mononucleosis. The results were compared with those obtained in 30 healthy adult volunteers and in 15 healthy children of the same age. Adhesiveness was significantly higher in adults than in healthy children, and significantly higher in healthy children than in children with viral infection. In 7 patients tested one month after regression of the disorder, PMN adhesiveness had returned to normal.