Nicole Schlegel

Intermittent hemophagocytic lymphohistiocytosis is a regular feature of lysinuric protein intolerance

We describe 4 cases of lysinuric protein intolerance, which all fulfilled the diagnostic criteria for hemophagocytic lymphohistiocytosis. Mature histiocytes and neutrophil precursors participated in hemophagocytosis in the bone marrow. Moreover, serum levels of ferritin and lactate dehydrogenase were elevated, hypercytokinemia was present, and soluble interleukin-2 receptor levels were increased up to 18.6-fold. The diagnosis of lysinuric protein intolerance should therefore be considered in any patient presenting with hemophagocytic lymphohistiocytosis.

Recombinant tissue-type plasminogen activator therapy of thrombosis in 16 neonates

We report 16 cases of neonatal vascular thrombosis treated with the same protocol for recombinant tissue-type plasminogen activator infusion. Flow restoration was complete in seven patients, partial in seven, and absent in two. Safety was satisfactory provided contraindications were respected.

Mutation spectrum and genotype-phenotype correlations in a large French cohort of MYH9-Related Disorders

MYH9‐Related Disorders are a group of rare autosomal dominant platelet disorders presenting as nonsyndromic forms characterized by macrothrombocytopenia with giant platelets and leukocyte inclusion bodies or as syndromic forms combining these hematological features with deafness and/or nephropathy and/or cataracts. They are caused by mutations in the MYH9 gene encoding the nonmuscle myosin heavy chain II‐A (NMMHC‐IIA). Until now, at least 49 MYH9 mutations have been reported in isolated cases or small series but only rarely in large series. We report the results of an 8‐year study of a large cohort of 109 patients from 37 sporadic cases and 39 unrelated families. We have identified 43 genetic variants, 21 of which are novel to our patients. A majority, 33 (76.7%), were missense mutations and six exons were preferentially targeted, as previously published. The other alterations were three deletions of one nucleotide, one larger deletion of 21 nucleotides, and one duplication. For the first time, a substitution T>A was found in the donor splice site of intron 40 (c.5765+2T>A). Seven patients, four from the same family, had two genetic variants. The analysis of the genotype‐phenotype relationships enabled us to improve the knowledge of this heterogeneous but important rare disease.

Normal ranges and genetic variants of antithrombin, protein C and protein S in the general Chinese population. Results of the Chinese Hemostasis Investigation on Natural Anticoagulants Study I Group

Background Inherited deficiency of antithrombin, protein C and protein S, three important, naturally occurring coagulation inhibitors, might play a major role in the occurrence of venous thromboembolism in Chinese. The establishment of age- and gender-related normal ranges of these inhibitors is crucial for an accurate diagnosis of these deficiencies. Design and Methods We designed a prospective cross-sectional study recruiting healthy adults from four university–affiliated hospitals in China. Antithrombin, protein C and protein S were studied by measuring their activity. Gene analysis was performed when natural anticoagulant deficiency was suspected. Polymorphisms of the factor V gene were searched for among subjects who were positive for activated protein C resistance. Results In 3493 healthy Chinese adults (1734 men, 1759 women; age 17–83 years), we found higher age-adjusted activities for protein C and protein S in men than in women but no sex difference for antithrombin. In women, mean protein C and protein S activities increased with age. In men, mean protein C levels increased with age up to the age of 49 but decreased after 50 years old; mean protein S levels decreased after 50 years of age. Antithrombin levels remained stable over time in women but decreased significantly after 50 years of age in men. Reference values according to age and sex allowed the identification of 15 genetic variants (protein C: 10, antithrombin: 3, protein S: 2) in subjects with protein activity below the 1st percentile. Conclusions This is the largest survey ever conducted in the healthy general Chinese population. These normal ranges provide the essential basis for the diagnosis and treatment of thrombosis in Chinese.

Effect of Individual Plasma Lipoprotein(a) Variations In Vivo on Its Competition With Plasminogen for Fibrin and Cell Binding An In Vitro Study Using Plasma From Children With Idiopathic Nephrotic Syndrome

Simultaneous natural changes in lipoprotein(a) [Lp(a)] and plasminogen occur in the nephrotic syndrome and offer a unique opportunity to investigate their effects on plasminogen activation under conditions fashioned in vivo. Plasminogen, Lp(a), and apolipoprotein(a) in plasma were characterized, and their competitive binding to carboxy-terminal lysine residues of fibrin and cell membrane proteins was determined in nephrotic children during a flare-up of the disease (61 cases) and after 6 weeks (33 cases) and 6 months (42 cases) of remission. Low plasminogen concentrations (median 1.34 micromol/L, range 0.39 to 1.96 micromol/L) and high Lp(a) levels (median 0.27 g/L, range 0.07 to 2. 57 g/L) were detected at flare-up. These changes were associated with an increased Lp(a) binding ratio onto fibrin (3.13+/-0.48) and cells (1.53+/-0.24) compared with binding ratios of control children (1.31+/-0.19 and 1.05+/-0.07, respectively) with normal plasminogen and low Lp(a) (median 0.071 g/L). After 6 weeks and 6 months of remission, the values for net decrease in Lp(a) binding to fibrin were 1.7+/-0.22 (after 6 weeks) and 1.88+/-0.38 (after 6 months) and were correlated with low Lp(a) concentrations (median 0.2 g/L, range 0.07 to 0.8 g/L; and median 0.12 g/L, range 0.07 to 1.34 g/L) and inversely associated with increased plasminogen levels (median 1.82 micromol/L, range 1.4 to 2.1 micromol/L; and median 1.58 micromol/L, range 1.1 to 2.1 micromol/L). These studies provide the first quantitative evidence that binding of Lp(a) to lysine residues of fibrin and cell surfaces is directly related to circulating levels of both plasminogen and Lp(a) and that these glycoproteins may interact as competitive ligands for these biological surfaces in vivo. This mechanism may be of relevance to the atherothrombotic role of Lp(a), particularly in nephrotic patients.

Immunogenicity of hepatitis B vaccine (HEVAC B) in children with advanced renal failure

The immune response after hepatitis B (HB) vaccine HEVAC B was studied in 33 children (mean age 10±4 years) with advanced renal failure. Responders and protected patients were defined by antibody titres to HB surface antigen (anti-HBs) of greater than 10 and 50 mIU/ml, respectively. All received the initial recommended three injections at monthly intervals, and 23 received a booster injection (IB) 11±1 months after the third injection (I3). Loss of protection after I3 led to additional injections in 8 patients (25%). Vaccine was well tolerated and no HB infection occurred during the follow-up period (19±10 months). The percentage of responders was 91% 2±1 months after I3, and 100% 1 month, 13±1 months and 26±2 months after IB. The percentages of protected patients at these dates were 91%, 95%, 100% and 100%. Anti-HBs titres 1–3 months after I3 were useful for indicating those patients likely to have a rapid decline in anti-HBs titres, thus requiring serial anti-HBs determinations and additional injections to prevent a loss of protection. We conclude that, at the expense of a reinforced vaccination schedule in 25% of patients, HEVAC B vaccine can safely achieve a sustained protection in more than 90% of uracmic children.

Urokinase treatment of pulmonary artery thrombosis complicating the pediatric nephrotic syndrome

Two pediatric patients with severe pulmonary thrombosis complicating a lipoid nephrosis were treated with urokinase administered either as a continuous infusion or in massive bolus doses. Both patients recovered but one died suddenly 2 yr later after recurrence of the nephrotic syndrome and probably new massive pulmonary thrombosis. These patients had severe abnormalities of hemostasis and fibrinolysis, which favored thrombosis and complicated fibrinolytic treatment.

Absence of plasma prostacyclin stimulating activity deficiency in hemolytic uremic syndrome

We compared the effect of plasma from 19 children with hemolytic uremic syndrome (HUS) on prostacyclin (PGI2) production by fresh rat aortic rings to the effect of plasma from 17 age- and sex-matched normal children, taking into account the PGI2 baseline aortic production (PGI2 release in presence of buffer, 21 determinations). After 10, 20, 30, 40, and 60 minutes incubation of rat aortic tissue with either plasma or buffer, the presence of PGI2 was studied by measuring by radioimmunoassay (RIA) the concentration of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha). 6-keto-PGF1 alpha production increased with time in the two groups of plasma samples and in the presence of buffer, but 6-keto-PGF1 alpha production (ng/mg dried tissue) after 30 minutes incubation and mean 6-keto-PGF1 alpha production (slope of regression line, ng/mg/min) were significantly (P less than 0.01) lower in the presence of normal plasma compared with buffer, and significantly (P less than 0.01) higher in the presence of HUS plasma compared with normal plasma. There was no significant difference between buffer and HUS plasma. We conclude that, under our experimental conditions, normal plasma had an inhibitory activity on 6-keto-PGF1 alpha production by rat aorta. This inhibitory activity was absent in HUS plasma.

Platelet morphology analysis.

Platelets are very small blood cells (1.5-3 μm), which play a major role in primary haemostasis and in coagulation mechanisms. Platelet characterization requires their counting (see Chapter 15 ) associated with accurate morphology analysis. We describe the major steps in order to correctly obtain stained blood films, which can be analyzed by optical microscope. Platelet morphology abnormalities are found in acquired malignant hematological diseases such myeloproliferative or myelodysplastic syndromes and acute megakaryoblastic leukemia. A careful analysis of the platelet size and morphology, by detecting either normal platelets with or without excessive anisocytosis, microplatelets, or large/giant platelets, will contribute to inherited thrombocytopenia diagnosis and gather substantial data when looking for an acquired platelet disorders.

Apparent homozygosity for p.E1841K mutation in a patient with MYH9-related disorder: a misdiagnosis for an autosomal dominant disorder?

To the Editor: Poopak B. et al. have reported (Eur J Haematol, 2011, 86(4):357) the case of a patient (P1) with MYH9-related disorder (MYH9-RD) that they have considered to be homozygous for p.1841E>K mutation in the non-muscle myosin heavy chain IIA (NMMHC-IIA). The homozygosity of P1, father of a 15-year-old boy (P2) heterozygous for p.1841E>K, was assessed by DNA sequence analysis. As all patients published with MYH9-RD, an autosomal dominant disorder, are heterozygotes, such a result deserves to be demonstrated by unquestionable data in order to confirm the results of the chromatogram shown by Poopak et al. Several causes of false homozygosity should have been examined. One of the most frequent of these causes is the inhibition or absence of PCR amplification of one allele. Could the authors precise the sequences of the primers used and their corresponding positions on MYH9 gene? Did they check the sequencing result using several couples of different primers? Did they control their result with several different Taq polymerases, to exclude possible error because of this enzyme? Did they analyse in detail the sequence of exon 38 and its junctions with introns 37 and 38, to detect regions difficult to amplify or containing SNPs and possibly preventing the hybridisation of the primers with the patient’s DNA? We have analysed the MYH9 gene in a cohort of 109 MYH9-RD patients with mutations in the MYH9 gene. We have had to face a problem of false homozygosity in exon 1 of MYH9 gene for 5 patients (Fig. 1), because of the presence of a poly G repeat in intron 1 preventing the PCR amplification of exon 1. If the primer was located downstream of the poly G repeat, only five samples harboured a mutation, two heterozygous, but three homozygous. If the primer was located upstream of the poly G repeat, seven samples harboured a mutation, heterozygous in the seven cases. These examples show that if one of the two alleles is not amplified, a heterozygous patient may be found either homozygous for the mutation (amplification of the mutated allele only) or normal (amplification of the normal allele only).