ALBERT

Description: Congenital deficiency of factor XI is a rare condition associated with a mild to moderate bleeding diathesis that is most commonly found in persons of Jewish ancestry. The disorder has been reported sporadically in a number of other ethnic groups, but rarely in the black population. We report on the genetic analysis of the factor XI genes of two African American patients: a 9-year-old boy (the propositus) with mild factor XI deficiency and his mother. Both individuals have lifelong histories of excessive bleeding. Dideoxyfingerprinting, a technique combining components of single-strand conformational polymorphism analysis and dideoxy-chain termination sequencing, was used in the analysis. Both patients were found to be heterozygous for a mutation changing serine 248 to glutamine, whereas the propositus was heterozygous for an additional mutation on the paternal allele changing glutamine 226 to arginine. Both mutations reside in the third apple domain of the factor XI heavy chain, an area that has been shown to contain binding sites for factor IX, platelets, and glycosaminoglycans. Previously reported mutations in the factor XI gene seem to cause deficiency primarily by reducing protein expression. Because both alleles in the propositus contain amino acid substitutions, the significant amount of circulating factor XI in his plasma must be comprised entirely of abnormal molecules. Factor XI circulates as a homodimer, and the presence of mutations in both alleles of the factor XI gene suggests that his bleeding disorder is caused in part by the effect of the two abnormal gene products forming dimers in different combinations. Three neutral (not associated with amino acid changes) DNA polymorphisms were also identified in the two subjects: a C to T change at nucleotide 472 in exon 5, A to G at nucleotide 844 in exon 8, and T to C at nucleotide 1234 in exon 11. Analysis of a random sample of normal volunteers showed that these polymorphisms are relatively common, with allele frequencies of 7.4%, 19%, and 18%, respectively. This suggests that there is considerable genetic heterogeneity in the factor XI gene. © 1998 by The American Society of Hematology.

Description: Congenital deficiency of factor XI is a rare condition associated with a mild to moderate bleeding diathesis that is most commonly found in persons of Jewish ancestry. The disorder has been reported sporadically in a number of other ethnic groups, but rarely in the black population. We report on the genetic analysis of the factor XI genes of two African American patients: a 9-year-old boy (the propositus) with mild factor XI deficiency and his mother. Both individuals have lifelong histories of excessive bleeding. Dideoxyfingerprinting, a technique combining components of single-strand conformational polymorphism analysis and dideoxy-chain termination sequencing, was used in the analysis. Both patients were found to be heterozygous for a mutation changing serine 248 to glutamine, whereas the propositus was heterozygous for an additional mutation on the paternal allele changing glutamine 226 to arginine. Both mutations reside in the third apple domain of the factor XI heavy chain, an area that has been shown to contain binding sites for factor IX, platelets, and glycosaminoglycans. Previously reported mutations in the factor XI gene seem to cause deficiency primarily by reducing protein expression. Because both alleles in the propositus contain amino acid substitutions, the significant amount of circulating factor XI in his plasma must be comprised entirely of abnormal molecules. Factor XI circulates as a homodimer, and the presence of mutations in both alleles of the factor XI gene suggests that his bleeding disorder is caused in part by the effect of the two abnormal gene products forming dimers in different combinations. Three neutral (not associated with amino acid changes) DNA polymorphisms were also identified in the two subjects: a C to T change at nucleotide 472 in exon 5, A to G at nucleotide 844 in exon 8, and T to C at nucleotide 1234 in exon 11. Analysis of a random sample of normal volunteers showed that these polymorphisms are relatively common, with allele frequencies of 7.4%, 19%, and 18%, respectively. This suggests that there is considerable genetic heterogeneity in the factor XI gene. © 1998 by The American Society of Hematology.

Description: The aim of this study was to identify the molecular mechanisms involved in neutrophil adhesion to immobilized platelets with particular focus on the possible existence of a juxtacrine system for neutrophil-platelet interactions. Platelets were immobilized onto collagen (type I)-coated coverslips that were placed in a flow chamber and neutrophils were perfused across these confluent monolayers at a shear stress of 1 to 4 dynes/cm2. Neutrophils rolled, and a significant proportion (25% to 50%) adhered to platelet monolayers. P-selectin was expressed in very large quantities on the surface of platelets and mediated all of the rolling, whereas the β2-integrin mediated firm adhesion. An activation mechanism for adhesion was necessary inasmuch as fixed neutrophils continued to roll on immobilized platelets, but did not adhere. Platelets adherent to collagen produced significant levels of platelet-activating factor (PAF). Accordingly, the firm adhesion of neutrophils to platelets was significantly inhibited by a PAF receptor antagonist (WEB 2086). Treatment of only the platelets with acetylhydrolase, which converts membrane-associated PAF to lyso-PAF, prevented 60% of the adhesion. These data suggest that PAF, on the surface of platelets, mediated a significant portion of the adhesive interaction. Addition of some selectin-binding carbohydrates (fucoidan or soluble SLEx analogs but not dextran sulfate) to the platelets caused rolling neutrophils to immediately adhere, an event that was not observed on histamine or thrombin-treated endothelium or P-selectin transfectants. These data support the view that a juxtacrine activation process exists on immobilized platelets for neutrophils. This process can be greatly enhanced on platelets and may involve a signaling mechanism through P-selectin.

Description: Epstein-Barr virus–induced lymphoproliferative disease (EBV-LPD) is a serious and potentially fatal complication after allogeneic stem cell transplantation (SCT). To evaluate levels of EBV DNA in SCT patients, a semiquantitative polymerase chain reaction (PCR) assay was developed. DNA was extracted from peripheral blood leukocytes and diluted, and PCR was performed by using a primer set specific for a well-conserved sequence of the internal repeat 1 region of the EBV genome. Forty-one SCT patients were screened with this method. Thirty-seven patients received allogeneic transplants, of which 18 were T-cell–depleted marrow. Four additional patients received autologous SCT, one of which was T-cell depleted. The mean time of follow-up by EBV PCR was 147 days (range, 47 to 328 days) posttransplant. The range of EBV copies/μg DNA from normal EBV sero-positive donors was 40 to 4,000. Seven patients had ≥40,000 copies of EBV DNA/μg DNA, all of whom were recipients of T-cell–depleted SCT. Five of the seven patients with elevated levels of EBV DNA developed EBV-LPD. Four of these five patients with EBV-LPD had elevated levels of EBV DNA from 1 to 8 weeks before diagnosis. Two patients with EBV-LPD had normal levels of EBV DNA, and two patients with ≥40,000 copies EBV/μg DNA did not develop EBV-LPD. In one patient, clinical resolution of disease correlated with a decrease in EBV DNA and an increase in the level of EBV-specific cytotoxic T-cell precursors. These data indicate that the measurement of EBV viral load with semiquantitative PCR is useful in detecting EBV-LPD in high-risk patients before the onset of clinical symptoms. Because not all patients with elevated levels of EBV DNA develop EBV-LPD, semiquantitative PCR results cannot substitute for clinical, radiographic, and pathological confirmation of this diagnosis.

Description: Epstein-Barr virus–induced lymphoproliferative disease (EBV-LPD) is a serious and potentially fatal complication after allogeneic stem cell transplantation (SCT). To evaluate levels of EBV DNA in SCT patients, a semiquantitative polymerase chain reaction (PCR) assay was developed. DNA was extracted from peripheral blood leukocytes and diluted, and PCR was performed by using a primer set specific for a well-conserved sequence of the internal repeat 1 region of the EBV genome. Forty-one SCT patients were screened with this method. Thirty-seven patients received allogeneic transplants, of which 18 were T-cell–depleted marrow. Four additional patients received autologous SCT, one of which was T-cell depleted. The mean time of follow-up by EBV PCR was 147 days (range, 47 to 328 days) posttransplant. The range of EBV copies/μg DNA from normal EBV sero-positive donors was 40 to 4,000. Seven patients had ≥40,000 copies of EBV DNA/μg DNA, all of whom were recipients of T-cell–depleted SCT. Five of the seven patients with elevated levels of EBV DNA developed EBV-LPD. Four of these five patients with EBV-LPD had elevated levels of EBV DNA from 1 to 8 weeks before diagnosis. Two patients with EBV-LPD had normal levels of EBV DNA, and two patients with ≥40,000 copies EBV/μg DNA did not develop EBV-LPD. In one patient, clinical resolution of disease correlated with a decrease in EBV DNA and an increase in the level of EBV-specific cytotoxic T-cell precursors. These data indicate that the measurement of EBV viral load with semiquantitative PCR is useful in detecting EBV-LPD in high-risk patients before the onset of clinical symptoms. Because not all patients with elevated levels of EBV DNA develop EBV-LPD, semiquantitative PCR results cannot substitute for clinical, radiographic, and pathological confirmation of this diagnosis.