The hemoglobin (Hb) variants represent a well-documented array (>800 in number) of amino acid substitutions, hybrid Hbs and Hbs with extended/shortened amino acid sequences. The majority of these structural alterations have little or no impact on the well being of the individual. However, some can result in severe disease when inherited in a homozygous or compound heterozygous state (e.g. sickle cell disease), while others can give a thalassaemic picture if the globin variant is highly unstable. Kings’s College Hospital (KCH), which is a referral centre for Hb variants, uses automated HPLC (Biorad Variant II) as the first line screening test, followed by further electrophoretic separations (isoelectric focusing (IEF), cellulose acetate membrane (CAM) and acid gel electrophoresis (AGE)) if a variant is present. Definitive diagnosis is then achieved by either mass spectrometry or DNA analysis of the suspected globin gene. The former method is quick and uses small quantities of blood but the equipment is expensive and requires a high degree of expertise, making it unavailable to many institutions. PCR based DNA diagnostics is routine in many service pathology laboratories but it is hampered by the cost of consumables and the time it takes. To minimise these KCH has developed a database that statistically evaluates the phenotypic separations and predicts which Hb variant is present based on past data. A single entry from any one of the tests (HPLC, IEF, CAM, AGE or mass difference) is enough to generate a prediction. Using this prediction only a small region of the putative globin gene will need sequencing or analysis by restriction digest, thereby reducing costs and turnaround times. The database contains the phenotypic separation data of >700 samples collected over six years. Variants were definitively diagnosed using electrospray ionisation mass spectrometry, complemented in some cases by DNA analysis. KCH will continue to enter variant data to increase the data set and improve the tool’s accuracy and aims to make the prediction tool available online so that other laboratories may obtain predictions and add their data. Laboratories will then be able to retrieve predictions based on their own past data or the entire database.
Extensive molecular studies have characterized 15 dimorphic and 2 multiallelic genetic markers within the human alpha-globin gene cluster. Analysis of these markers in 9 populations has shown that the alpha-globin locus is remarkably polymorphic and is therefore an ideal marker on chromosome 16 for the construction of a human genetic linkage map. The combined analysis of 9 polymorphic markers has established alpha-globin haplotypes that provide the means to study the molecular genetics and common mutants of this cluster. The novel association of a conventional restriction fragment length polymorphism haplotype and linked, hypervariable regions of DNA should allow a comparison of the rate of change of such markers.
Antithrombin is the most important physiological proteinase inhibitor of thrombin and other coagulation proteinases. It is a single chain glycoprotein of MW 58 200 which has sequence homology with α1-antitrypsin and other members of the serpin superfamily of inhibitors. Two functional domains of importance have been identified, the reactive centre that interacts with the proteinase and a heparin binding domain. Failure to maintain an adequate level of functional antithrombin in plasma results in an increased risk of thromboembolism: deficiency can be inherited or acquired. There is still uncertainty regarding the prevalence of inherited deficiency and the prevalence of thrombosis in affected individuals. The production of antithrombin is under the control of a single gene which is localized on chromosome 1q 23–25. Characterization of the coding sequence, which is distributed over seven exons, has allowed the analysis of the molecular basis for inherited antithrombin deficiency. To date more than 100 cases have been successfully investigated at the gene and/or protein sequence level and 40 novel mutations have been identified. Mutations causing amino acid substitutions solely affecting the heparin binding site have thus far been located primarily at the N-terminal region of the molecule, residues 7–129; this region has been postulated to align as a positive groove in the molecule that forms the primary contact region for the essential antithrombin binding pentasaccharide of heparin. Not all the residues in which substitutions have been found are basic and some serve to maintain the conformation of nearby basic regions. Examples of this are provided by the Pro-41 to Leu mutation and a recently investigated mutant, Leu-99 to Phe. The reactive site defects are an interesting group, including those that alter P1, P1' and P12-P10 residues. Perhaps more remote mutations can also be included such as Pro-429 to Leu. The P1 and P1' mutations directly block interaction of the proteinase with antithrombin, while P12-P10 mutants (which have mutations affecting serpin strand s4A) enable the substrate reaction to proceed to completion, i.e. the antithrombin-thrombin complex is not stabilized and the mutant inhibitor is transformed into a substrate. The effect of the Pro-429 to Leu substitution is impairment of the reactive site and heparin binding, and the finding that this variant is not completely recognized by some MAbs implies a conformational change at the C terminus. Another group (nine cases) of interesting mutations is emerging, that has its primary defect in or near serpin strand 1C, amino acid sequence 402–407. These mutations are adjacent to an invariant region in the serpin family that appears to be important for their overall structure. The identified amino acid substitutions in antithrombin produce pleiotropic effects on the inhibitor, altering the reactive site, heparin binding properties and in many cases, the plasma level of protein. Appreciable progress is now also being made on the identification of cases of 'classical' antithrombin deficiency. While a few partial gene deletions are currently being characterized, most cases that have been identified are point mutations (including small deletions and insertions) in the coding region. There is one common mutation at position 129 (Arg to Stop, six cases), a small number of mRNA splice site abnormalities and the remainder are frameshifts or critical amino acid sequence changes distributed throughout the coding region of antithrombin.
We report prenatal diagnosis in a family with combined antithrombin deficiency (type II heparin binding site) and factor V 506 Arg to Gln mutation. Both clinically unaffected parents are heterozygous for the antithrombin mutation, which results in a 99 Leu to Phe substitution, and the father is also heterozygous for the factor V gene defect. There is one daughter, homozygous for the antithrombin and heterozygous for the factor V mutations, who suffered a right‐sided hemiparesis at the age of 4 months due to occlusion of the left middle cerebral artery and a large left sided infarct followed by further thromboembolic events. The family requested prenatal diagnosis and chorionic villi was sampled at 12 weeks gestation. The fetus was shown to be heterozygous for the antithrombin and factor V gene mutations, the same genotype as the unaffected father. No further intervention was considered necessary. To our knowledge this is the first report of prenatal diagnosis in antithrombin deficiency.
HbSC disease is the second commonest form of sickle cell disease, with poorly understood pathophysiology and few treatments. We studied the role of K-Cl cotransport activity in determining clinical and laboratory features, and investigated its potential role as a biomarker. Samples were collected from 110 patients with HbSC disease and 41 with sickle cell anemia (HbSS). K-Cl cotransport activity was measured in the oxygenated (K-Cl cotransport100) and deoxygenated (K-Cl cotransport0) states, using radioactive tracer studies. K-Cl cotransport activity was high in HbSC and decreased significantly on deoxygenation. K-Cl cotransport activity correlated significantly and positively with the formation of sickle cells. On multiple regression analysis, K-Cl cotransport increased significantly and independently with increasing reticulocyte count and age. K-Cl cotransport activity was increased in patients who attended hospital with acute pain in 2011 compared to those who did not (K-Cl cotransport100: mean 3.87 versus 3.20, P=0.009, independent samples T-test; K-Cl cotransport0: mean 0.96 versus 0.68, P=0.037). On logistic regression only K-Cl cotransport was associated with hospital attendance. Increased K-Cl cotransport activity was associated with the presence of retinopathy, but this effect was confounded by age. This study links variability in a fundamental aspect of cellular pathology with a clinical outcome, suggesting that K-Cl cotransport is central to the pathology of HbSC disease. Increased K-Cl cotransport activity is associated with increasing age, which may be of pathophysiological significance. Effective inhibition of K-Cl cotransport activity is likely to be of therapeutic benefit.
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