Mendelian diseases among Roman Jews: implications for the origins of disease alleles.

The Roman Jewish community has been historically continuous in Rome since pre-Christian times and may have been progenitor to the Ashkenazi Jewish community. Despite a history of endogamy over the past 2000 yr, the historical record suggests that there was admixture with Ashkenazi and Sephardic Jews during the Middle Ages. To determine whether Roman and Ashkenazi Jews shared common signature mutations, we tested a group of 107 Roman Jews, representing 176 haploid sets of chromosomes. No mutations were found for Bloom syndrome, BRCA1, BRCA2, Canavan disease, Fanconi anemia complementation group C, or Tay-Sachs disease. Two unrelated individuals were positive for the 3849 + 10C->T cystic fibrosis mutation; one carried the N370S Gaucher disease mutation, and one carried the connexin 26 167delT mutation. Each of these was shown to be associated with the same haplotype of tightly linked microsatellite markers as that found among Ashkenazi Jews. In addition, 14 individuals had mutations in the familial Mediterranean fever gene and three unrelated individuals carried the factor XI type III mutation previously observed exclusively among Ashkenazi Jews. These findings suggest that the Gaucher, connexin 26, and familial Mediterranean fever mutations are over 2000 yr old, that the cystic fibrosis 3849 + 10kb C->T and factor XI type III mutations had a common origin in Ashkenazi and Roman Jews, and that other mutations prevalent among Ashkenazi Jews are of more recent origin.

Differentiation of autoimmune thrombocytopenia from thrombocytopenia associated with immune complex disease: systemic lupus erythematosus, hepatitis-cirrhosis, and HIV-1 infection by platelet and serum immunological measurements.

A method and approach are described to differentiate classic autoimmune thrombocytopenia (ATP) from immune complex-associated thrombocytopenia in systemic lupus (SLE), hepatitis/chronic liver disease (LIV-ITP) and HIV-1 related thrombocytopenia (HIV-1-ITP). The platelet immunologic profile of IgG, C3C4 and IgM was measured with a solid-phase ELISA, employing 125I-staphylococcal protein A to detect indicator antibody binding. Polyethylene glycol was employed to precipitate immune complexes (PEG-IC). Platelet-associated IgG (PAIgG) was 2.8-, 5.6- and 5.8-fold higher in SLE, LIV-ITP and HIV-1-ITP patients respectively compared to ATP patients: platelet C3C4 was 3.2-, 4.8- and 4.5-fold higher respectively; platelet IgM was 2.2-, 3.7- and 3.8-fold higher respectively; serum PEG-IC levels were 4.2-, 4.8- and 2.1-fold higher respectively. With all parameters measured, there was no overlap between the 75th percentile for ATP patients and the 25th percentile for all three cohorts. The likelihood of having a platelet C3C4 level higher than the highest ATP level was 69% for SLE, 90% for LIV-ITP and 94% for HIV-1-ITP respectively; with PEG-IC measurements the likelihood was 83%, 100% and 100% respectively. Serum IgG, C3, C4, IgM and PEG-IC were examined for a possible relationship with platelet measurements. Except for a positive correlation between serum and platelet IgM in ATP, r = 0.5, P < 0.04, there was no positive correlation with any of the parameters measured. An inverse correlation was noted between PEG-IC level and platelet C3C4 in SLE, r = 0.7, P < 0.04. Thus platelet immunologic profile and serum PEG-IC level measurements differentiated classic ATP from immune complex-associated thrombocytopenias (SLE, LIV-ITP, HIV-1-ITP). Except for IgM measurements in ATP, platelet measurements could not be attributed to their respective serum concentration.

Severe factor XI deficiency in an Arab family associated with a novel mutation in exon 11.

We investigated an 8-year-old Arab girl with severe factor XI deficiency; one sibling and her father also have severe factor XI deficiency. Her parents and her father's parents are first cousins. Restriction analysis and DNA sequencing excluded the type I, II, III and IV mutations. We demonstrated a previously undescribed C-->A mutation at nucleotide 1254 in exon 11 resulting in a threonine to asparagine (T-->N) substitution at amino acid 386. We postulate that this substitution interferes with folding and secretion of the molecule.

Prothrombin synthesis in the adult and fetal liver.

Plasma levels of blood coagulation zymogens are lower in the newborn than in the adult, with the lowest levels being in preterm infants. It is not known if the lower coagulation factor levels reflect differences in synthesis, secretion or catabolism. Using a rabbit model we have compared prothrombin synthesis in the fetus and adult. In previous studies we attempted to compare transcription in the adult and fetal liver by extraction of mRNA, immobilization on a membrane and hybridization with a labeled cDNA for rabbit prothrombin. Comparison was impaired by the markedly dissimilar composition of fetal and adult rabbit liver; fetal liver is approximately fifty percent hematopoietic tissue even at term (1). In the present study, to obtain a more meaningful comparison we have employed in situ hybridization to compare directly prothrombin expression in adult and fetal liver. We report here that fetal liver contains more prothrombin mRNA than does adult liver. We have further compared prothrombin levels in protein extracts of adult and fetal liver and found that per microgram of extract, fetal liver contains as much prothrombin as does the adult. We conclude that the lower plasma prothrombin levels in the fetus do not reflect a lower rate of synthesis.

Thrombocytosis after major lower extremity trauma: mechanism and possible role in free flap failure.

Microvascular thrombosis and free flap failure are complications of free tissue transfer for coverage of lower extremity soft-tissue and bony defects despite appropriate vessel selection and adherence to meticulous technique. Increased rates of flap failure have been associated with reconstruction performed between 3 days and 6 weeks after injury, as well as in patients with thrombocytosis. We have found that serum platelet levels rise significantly after lower extremity injury. It is our theory that a circulating mediator or cytokine is released in response to injury, inducing the thrombocytosis. Twenty-one patients with Gustilo grade IIIb and IIIc injuries were studied prospectively. Serum was collected throughout the postinjury period. Platelet count, leukocyte count, hemoglobin concentration, and hematocrit were determined. Samples were also subjected to a platelet aggregation study as well as enzyme-linked immunosorbent assay for interleukin-3, interleukin-6, interleukin-11, and granulocyte macrophage-colony-stimulating factor. Megakaryocyte growth and development factor enzyme-linked immunosorbent assay and a myleoproliferative leukemia virus-transfected cell line assay for thrombopoietin were performed. Bone marrow was studied with flow cytometric analysis. Mean initial platelet count was 196,000 per cubic millimeter. There was an initial 26% decline to 140,000 per cubic millimeter, followed by an increase to 361% of baseline on day 16. No significant variations in serum leukocyte count or hemoglobin concentration were seen. Spontaneous and induced platelet aggregation responses were normal. Interleukin-6 was detected at elevated levels. However, interleukin-3, interleukin-11, granulocyte macrophage-colony-stimulating factor, and thrombopoietin were not measurable. Marked megakaryocytosis was seen on bone marrow analysis. Interleukin-6 may, therefore, play a role in the mechanism of thrombocytosis. We suggest that because patients with complex bony injuries of the leg experience platelet elevations that peak approximately 2 weeks after injury, microvascular free flap reconstructions should be considered high risk during this time period.

Transient hemorrhagic diathesis associated with an inhibitor of prothrombin with lupus anticoagulant in a 1 1/2-year-old girl: report of a case and review of the literature.

Acquired inhibitors of coagulation causing bleeding manifestations are rare in children, particularly without an associated underlying disorder such as autoimmune disease. We describe an otherwise healthy 1 1/2-year-old girl who had extensive spontaneous bruising and prolonged bleeding from venipuncture sites. Prothrombin time (PT) and activated partial thromboplastin time (APTT) were prolonged, with evidence of an immediate-acting inhibitor. Thrombin clotting time, fibrinogen, and platelets were normal. Biologic assay of factors II, V, VII, and X were all low, with increasing values at higher dilutions. However, by immunoassay and/or chromogenic assays, only factor II was reduced. An antibody which failed to neutralize prothrombin activity in vitro was detected against radiolabeled prothrombin. Coagulation studies normalized in parallel with clinical recovery and disappearance of the antibody. This case demonstrates acute hypoprothrombinemia-lupus anticoagulant syndrome as a rare presentation of bleeding diathesis in a healthy young child.

Functional activity of protein C in newborn infants. A report of a study and a review of the literature.

Protein C is lower in newborn infants than in adults. There are conflicting reports regarding functional activity and the presence of des-gamma carboxylated species in the newborn. We have compared protein C activity and antigenic level in newborn infants and found the activity: antigen ratio to be lower than in adults (0.69 versus 1.0). We discuss this finding in relation to the previously published reports.

Thrombocytopenia in children infected with human immunodeficiency virus: long-term follow-up and therapeutic considerations.

Among 180 children infected with human immunodeficiency virus (HIV-1), 14 (8%) developed thrombocytopenia during the course of the disease and have been followed for an average period of 18.8 months. Eight of 14 patients had clinical signs of bleeding. Increased levels of anti-platelet IgG antibodies were detected in 86% of patients tested and did not correlate with severity of disease. Eight patients were treated initially with intravenous immunoglobulins (IVIG) and responded with a transient increase in the platelet count of at least 30 x 10(9)/L. Sustained remission could not be achieved in the patients treated with IVIG alone. Corticosteroids were used in 6 patients who became refractory to IVIG and resulted in sustained remission in only one patient. Spontaneous remission of thrombocytopenia occurred in one patient. Ten patients were treated with zidovudine (ZVD) for a period of 3-20 months. Sustained improvement in the platelet counts occurred in only three of the children treated with ZVD.

Prothrombin expression in the adult and fetal rabbit liver.

Plasma prothrombin levels in newborn humans are lower than in adults. The same is true of many newborn and fetal mammals, including the rabbit. To determine if the lower levels are due to less expression of the protein, we have compared mRNA for prothrombin in fetal and adult rabbit liver. Northern blots were hybridized with a cDNA for rabbit prothrombin revealing a single mRNA of approximately 2 kb in both adult and fetal animals. mRNA specific for prothrombin was quantitated by slot blotting of RNA prepared from adults and fetuses aged 21 d to term (31 d). Prothrombin-specific mRNA in fetuses was greater than 50% of that in adults even when the fetal plasma prothrombin was only 15% of the adult level. This suggests that low plasma levels in the fetuses are not the result of less transcription. Examination of liver sections revealed that the predominant tissue in the fetus is hematopoietic, not hepatic. In the youngest fetuses, less than 20% of the liver consisted of hepatocytes, yet these fetuses expressed more than 50% of the adult level of prothrombin-specific mRNA. Thus, transcription of prothrombin mRNA may be proceeding at a greater rate in the fetal hepatocyte than in the adult, or hematopoietic cells may be expressing the protein. We conclude that in fetal rabbit liver, prothrombin is expressed at a high level relative to the hepatocyte content and that the cause of the low plasma levels is posttranscriptional.

Cloning and partial sequence of a cDNA for rabbit prothrombin.

A 1466 base pair cDNA for rabbit prothrombin has been isolated and partially sequenced. The deduced amino acid sequence shows considerable homology with the sequences of human and bovine prothrombin. The cDNA extends from the equivalent of nucleotide 516 in the bovine sequence through the coding region and 99 nucleotides in the 3' non-coding region.

Micromethod for demonstrating increased platelet surface immunoglobulin G: findings in acute, chronic, and human immunodeficiency virus-1-related immunologic thrombocytopenias.

A method has been developed for the demonstration of increased platelet surface IgG that uses 1 ml of blood regardless of the platelet count. Platelets are gel filtered to remove plasma and contaminating lymphocytes. They are then reacted with fluorescein-conjugated antihuman IgG and analysed by flow cytometry. Percent positive staining cells vary from 10% to 80% of total cells examined. A platelet antibody index is derived from the product of percent positive staining cells X mean fluorescence intensity of positive staining cells. All patients studied with chronic idiopathic thrombocytopenia purpura (ITP) or human immunodeficiency virus-1 (HIV-1)-related thrombocytopenia had increased platelet surface IgG. Twelve acute children and 11 chronic children had indices averaging 3.5- and 8.9-fold greater than 12 normal children, respectively. Five of 12 children with acute ITP had normal platelet IgG. There was no linear correlation between the platelet antibody index and platelet count. Platelets of patients with acute, chronic, or HIV-1-related ITP displayed autofluorescence. In chronic ITP, the percentage of platelets displaying autofluorescence had a significant negative correlation with the platelet count. This technique will be a valuable diagnostic tool in the pediatric population.

Protein S in the first year of life.

Total protein S, free protein S and C4b binding protein were measured in healthy term infants in order to establish the normal levels of these proteins during the first year of life. Total protein S rose from 36.5% of the adult mean level on day 1 of life to 90% between 6 and 12 months of age. Free protein S was 54.2% of the adult mean on day 1 of life, all values were in the adult range by 2 months and the mean value was no different from the adult at 4 months. This relatively high level of free and presumably active protein S reflects low levels of C4bBP at birth (28.8% of the adult mean) and a slow postnatal rise.

Effect of warfarin on prothrombin synthesis and secretion in human Hep G2 cells.

Prothrombin synthesis and secretion were studied in a human hepatoma cell line (Hep G2) incubated with 35S-methionine for 2 to 24 hours at 37 degrees C. Extracellular and intracellular prothrombin were detected by immunoprecipitation with affinity-purified antiprothrombin antibody. Incorporation of 35S-methionine into prothrombin was monitored by counting specific bands excised from 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Prothrombin represented 0.3% to 0.7% of total newly synthesized protein secreted into the media. Warfarin had no effect on total prothrombin synthesis (extracellular plus intracellular). However, warfarin inhibited secretion of newly synthesized prothrombin by 58% to 73% over a 2 to 4 hour period. This was accompanied by the intracellular accumulation of an immunoprecipitable species of prothrombin of 78 kd, 6 kd less than extracellular prothrombin. At the end of the 4-hour incubation with warfarin, intracellular prothrombin increased from 44% to 82% (twofold) of total prothrombin, whereas extracellular prothrombin decreased from 56% to 19% (threefold) of total prothrombin. After 24-hour incubation with warfarin, intracellular and extracellular immunoprecipitable prothrombin approached control values. Deglycosylation of extracellular and intracellular prothrombin with hydrofluoric acid (HF) resulted in a decrease in mol wt for both species to 66 kd. Endoglycosidase-H treatment, which digests "early mannosyl" residues, resulted in a decrease in the mol wt of the intracellular species of 8 kd with no effect on the extracellular species. Thus, the lower mol wt intracellular species that accumulates following early warfarin treatment is due to the presence of incompletely processed carbohydrate chain. The data are compatible with the hypothesis that optimum glycosylation and secretion require Vitamin K-dependent carboxylation.

An epidemic of maternal thrombocytopenia associated with elevated antiplatelet antibody. Platelet count and antiplatelet antibody in 116 consecutive pregnancies: relationship to neonatal platelet count.

Twenty-eight (24%) of 116 pregnant women studied prospectively during an 8-month period in 1983 had platelet counts of less than 150,000/mm3 at least once during pregnancy. Thirteen of these were thrombocytopenic in both the prenatal and the peripartum period. Eighteen were restudied 3 to 12 months after delivery. One woman, who was pregnant again, had a platelet count of 140,000/mm3. In the others, platelet counts were in the normal range. Platelet-associated immunoglobulin G and serum antiplatelet antibody levels were elevated in 79% and 61%, respectively, of these 28 women on at least one occasion. However, 59% of 73 pregnant nonthrombocytopenic women had increased platelet-associated immunoglobulin G levels and 59% had positive serum antiplatelet antibody test results. Twenty women who had increased platelet-associated immunoglobulin G levels and positive serum antiplatelet antibody test results were normal 6 to 10 months after delivery. Of 105 infants studied, 10 were thrombocytopenic. Neonatal thrombocytopenia was not predicted by maternal platelet count, platelet-associated immunoglobulin G, or serum antiplatelet antibody. By the fall of 1984, the incidence of thrombocytopenia had dropped to two in 280 consecutive pregnancies. We conclude that (1) epidemics of thrombocytopenia can occur in pregnant women and (2) if a women is found to be thrombocytopenic for the first time during pregnancy, she should not be subjected to the measures advocated for the management of pregnancy in women with autoimmune thrombocytopenic purpura.

Low protein C in the neonatal period.

Protein C was measured by electroimmunoassay in 47 infants within 24 h of delivery. Gestational age ranged from 28 to 43 weeks. The mean level was 27% (range less than 10-67%) of the normal adult mean. In the 22 infants who had no clinical problems, protein C levels correlated significantly with gestational age. In the 25 who were sick there was no correlation, and the mean level was significantly lower than that of the healthy infants. Postnatal rise was slow; on day 7 the mean was 32% and on day 28, 31%. Levels of protein C correlated significantly with prothrombin in both the healthy and sick infants. Crossed immunoelectrophoresis in the presence of calcium ions gave one protein C peak of the same electrophoretic mobility as is seen in plasma of healthy adults, indicating that the infants' protein C is gamma carboxylated. It is concluded that: (1) Protein C in neonates is in or below the range associated with thromboembolism in patients congenitally deficient in this protein; (2) protein C levels correlate with gestational age; and (3) the low levels during the neonatal period are not due to decreased gamma carboxylation but may reflect decreased synthesis when compared to the older child or the adult.