Jane M. Benson

F8 and F9 mutations in US haemophilia patients: correlation with history of inhibitor and race/ethnicity.

Summary.  Both genetic and treatment‐related risk factors contribute to the development of inhibitors in haemophilia. An inhibitor surveillance system piloted at 12 US sites has the goal of assessing risk factors through prospective data collection. This report examines the relationship of genotype and race/ethnicity to history of inhibitor in a large cohort of US haemophilia patients. Mutation analysis was performed on 676 haemophilia A (HA) and 153 haemophilia B (HB) patients by sequencing, Multiplex Ligation‐dependent Probe Amplification, and PCR for inversions in F8 introns 22 (inv22) and 1 (inv1). Two HB patients with deletions had history of inhibitor. In severe HA, frequency of history of inhibitor was: large deletion 57.1%, splice site 35.7%, inv22 26.8%, nonsense 24.5%, frameshift 12.9%, inv1 11.1% and missense 9.5%. In HA, 19.6% of 321 White non‐Hispanics (Whites), 37.1% of 35 Black non‐Hispanics (Blacks) and 46.9% of 32 Hispanics had history of inhibitor (P = 0.0003). Mutation types and novel mutation rates were similar across ethnicities. When F8 haplotypes were constructed, Whites and Hispanics showed only H1 and H2. Within H1, history of inhibitor was 12.4% in Whites, 40.0% in Blacks (P = 0.009) and 32.4% in Hispanics (P = 0.002). Inhibitor frequency is confirmed to vary by mutation type and race in a large US population. White patients with history of inhibitor did not exhibit rare F8 haplotypes. F8 gene analysis did not reveal a cause for the higher inhibitor frequencies in Black and Hispanic patients.

Prevalence of the prothrombin 20210 G-to-A variant in blacks: Infants, patients with venous thrombosis, patients with myocardial infarction, and control subjects☆☆☆

A genetic variation in the prothrombin gene is located in the 3-untranslated region at position 20210 where a G-->A transition occurs. The prevalence of the mutation is 1% to 2% in white populations, and the mutation is associated with an increased risk of venous thrombosis and myocardial infarction. We report the prevalence of the A allele in blacks at birth; in black control subjects with no history of heart attack, stroke, or blood clots; in black patients with venous thrombosis; and in black patients with myocardial infarction. Among 318 infants, the prevalence of the A allele was 0.2% (1 heterozygote), with an exact, one-sided upper 95% confidence limit of 0.7%. Among 185 control subjects the variant was absent, yielding an exact, one-sided upper 95% confidence limit of 0.8% for the A allele. The heterozygous genotype was found in 2 of 91 subjects with deep vein thrombosis and in none of 123 subjects with myocardial infarction. The very low prevalence of the A allele indicates that the prothrombin variant is not a major cause of venous thrombosis or myocardial infarction in blacks.

History and current status of newborn screening for hemoglobinopathies.

The impact of hemoglobinopathies on healthcare in the United States, particularly sickle cell disease (SCD), has been significant. Enactment of the Sickle Cell Anemia Control Act in 1972 significantly increased the federal interest in the SCDs and other hemoglobinopathies. Only since May 1, 2006, have all states required and provided universal newborn screening for SCD despite a national recommendation to this effect in 1987. In this article, we review the history of screening for SCD and other hemoglobinopathies, along with federal and state activities that have contributed to improved health outcomes for patients with SCD, as well as current newborn screening practices. We also chronicle the federal activities that have helped to shape and to refine laboratory screening and diagnostic proficiency. Finally, we review molecular testing strategies that have evolved and outline their possible future impacts on disease detection and outcome improvement.

Hormonal contraception, sickle cell trait, and risk for venous thromboembolism among African American women

OBJECTIVE We evaluated the effect of oral and other hormonal contraceptive (HC) use on venous thromboembolism risk among African American women and investigated whether the association was modified by the sickle cell trait. STUDY DESIGN We report the findings of a case-control study that included 60 African American women with an idiopathic, first episode of venous thromboembolism and 196 African American controls. RESULTS The odds of current HC use compared with noncurrent use contrasting cases and controls is 3.8 (95% confidence interval [CI], 1.7-8.1; P < .001). Among subjects with sickle cell trait, the odds ratio is higher (odds ratio [OR], 6.7; 95% CI, 1.0-43) than the odds ratio among subjects without sickle cell trait (OR, 2.6; 95% CI, 1.1-6.2), but the difference is not statistically significant. CONCLUSION This study provides persuasive evidence that hormonal contraceptive use increases venous thromboembolism risk among African American women and that the increase in risk may be larger among women with sickle cell trait.

Multiplex Analysis of Mutations in Four Genes Using Fluorescence Scanning Technology

Multiplex analysis of genetic mutations using fluorescence scanning methodology is an accurate, efficient, and cost-effective approach to genotypic characterization. Fluorescence labeling during the synthesis of polymerase chain reaction primers allows the application of this technology to well-established protocols. We have simultaneously analyzed the four polymorphisms of factor V Leiden (G1691A), prothrombin G20210A, 5,10-methylenetetrahydrofolate reductase C677T, and cystathionine beta-synthase 844ins68. Three of these mutations have been associated with an increased risk of thrombosis. Following polymerase chain reaction with fluorescence-labeled primers, the polymerase chain reaction products were digested with an appropriate restriction enzyme (if necessary for detection of the mutation), diluted into one tube per sample for co-loading (multiplex loading), and analyzed with GeneScan software for fragment analysis following capillary electrophoresis on an ABI PRISM 310 Genetic Analyzer (Foster City, CA, USA). Multiplex loading increased throughput without compromising precision.

A variant hemoglobin assicated with α-Thalassemia-2

Abstract Hb Evanston (α14 Trp → Arg) was detected on cellulose acetate at pH 8.4 as a band with an electrophoretic mobility similar to that of Hb S. In addition, a band migrating cathodic to Hb A2 suggested the presence of a variant Hb A2 with a substitution in the α-chain, a fact that was later confirmed by structural analysis. An unusual feature of Hb Evanston is its low percentage; less than 10% occurs in the hemolysate. Studies indicate that the variant is not unstable, but there appears to be a defect in globin-chain synthesis. Gene mapping also shows that it is associated with the α-thalassemia-2 gene. The variant has high oxygen affinity with normal cooperativity and a normal Bohr effect. The combination of Hb Evanston with α-thalassemia-2 produced anemia in this black family.

Fluorescent multiplex polymerase chain reaction analysis of four genes associated with inpaired fibrinolysis and myocardial infarction.

Multiplex polymerase chain reaction (PCR) allows for the simultaneous amplification of several genes, thereby optimizing the use of reagents and decreasing personnel time. Multiplex PCR was used to amplify four genes in one PCR reaction, demonstrating the advantage of multiplex PCR for our study since it allowed us to amplify four separate genes using only 1 μl DNA, thus maximizing the use of study DNA. As compared with conventional multiplex PCR analysis with ethidium bromide, incorporating fluorescence-labeled primers into multiplex PCR reactions facilitated accurate, simultaneous analysis of many DNA fragments within one base discrimination. We have used this fluorescence methodology to analyze polymorphisms associated with either impaired fibrinolysis or myocardial infarction. These include the angiotensin converting enzyme insertion/deletion (I/D) polymorphism in intron 16 of the DCP1 gene, the Alu I/D polymorphism of the tissue plasminogen activator-25 locus in intron 8, the plasminogen activator inhibitor 4G/5G repeat polymorphism, and the variable number tandem repeat of the endothelial cell nitric oxide synthase gene, all characterized by an insertion, deletion, or repeat. The amplified products were diluted 1 : 60 and analyzed on the ABI PRISM® 310 Genetic Analyzer using GeneScan® software. With this method, we were able to amplify four genes using 75% less reagents and personnel time, thus demonstrating the benefit of multiplex PCR and fluorescence technology.