Detection of fetal mutations causing hemoglobinopathies by non-invasive prenatal diagnosis from maternal plasma.

BACKGROUND: Prenatal diagnosis of hemoglobinopathies enables couples at risk to have a healthy child. Currently used fetal sampling procedures are invasive with some risk of miscarriage. A non-invasive approach to obtain fetal deoxyribonucleic acid (DNA) for diagnosis would eliminate this risk. AIM: To develop and evaluate a non-invasive prenatal diagnostic approach for hemoglobinopathies using cell-free fetal DNA circulating in the maternal plasma. SETTINGS AND DESIGN: Couples referred to us for prenatal diagnosis of hemoglobinopathies where the maternal and paternal mutations were different were included in the study. MATERIALS AND METHODS: Maternal peripheral blood was collected at different periods of gestation before the invasive fetal sampling procedure was done. The blood was centrifuged to isolate the plasma and prepare DNA. A size separation approach was used to isolate fetal DNA. Nested polymerase chain reaction (PCR)-based protocols were developed for detection of the presence or absence of the paternal mutation. RESULTS AND CONCLUSIONS: There were 30 couples where the parental mutations were different. Of these, in 14 cases the paternal mutation was absent and in 16 cases it was present in the fetus. Using cell-free fetal DNA from maternal plasma, the absence of the paternal mutation was accurately determined in 12 of the 14 cases and the presence of the paternal mutation was correctly identified in 12 of the 16 cases. Thus, this non-invasive approach gave comparable results to those obtained by the conventional invasive fetal sampling methods in 24 cases giving an accuracy of 80.0%. Although the nested PCR approach enabled amplification of small quantities of cell-free DNA from maternal plasma at different periods of gestation after size separation to eliminate the more abundant maternal DNA, an accurate diagnosis of the presence or absence of the paternal mutation in the fetus was not possible in all cases to make it clinically applicable.

Effect of radiation therapy on the latest generation of pacemakers and implantable cardioverter defibrillators: A systematic review.

The increasing human lifespan and development of technology over the last number of decades has seen an increase in the number of pacemaker and implantable cardioverter defibrillator (ICD) implantations worldwide. Given the number of risk factors common to both heart disease and cancer, it is not uncommon for several of these patients to present for radiation therapy treatment each year. A systematic review was conducted using online databases Medline and Scopus. Results were grouped into in vitro and in vivo studies. In 1994, the American Association of Physicists in Medicine (AAPM) defined guidelines for the management of these patients, which have since been adopted by many radiation oncology departments internationally. More recently, a number of studies have reported an increase in radiation sensitivity of these devices (encompassing the coiled metal leads and generator unit) due to the incorporation of complementary metal oxide semiconductor circuitry. Further avenues of device failure, such as the effect of dose rate and scatter radiation, have only more recently been investigated. There are also the unexplored avenues of electromagnetic interference on devices when incorporating newer treatment technologies such as respiratory gating and intensity modulated radiation therapy. It is suggested that each radiation oncology department employ a policy for the management of patients with ICDs and pacemakers, potentially based upon an updated national or international standard similar to that released by the AAPM in 1994.

Evaluation of a capillary blood collection system for screening for hemoglobinopathies in remote areas.

Accurate estimation of hemoglobin (Hbs) A, Hb A(2), Hb F and abnormal Hb is required for diagnosis of hemoglobinopathies and genetic counseling. High pressure liquid chromatography (HPLC) is the most suitable approach available. But for 70% of the rural Indian population, HPLC analysis facilities are not available and screening would require transportation of samples to laboratories in bigger cities. We thus evaluated the feasibility of using a kit designed for measuring Hb A(1c) using capillary blood for collection and preservation of samples over a period of 15 days at different temperatures for screening for hemoglobinopathies. Capillary blood (5 microl) of 90 individuals was collected in the capillary collection system and run on the Variant Hemoglobin Testing System on days 1, 3, 5, 8, 12 and 15 after incubation at 4, 22, 37, 42 and 50 degrees C. The stability of different Hbs varied at different temperatures. The stability was maintained for 12 to 15 days by most of the samples up to 37 degrees C. Hb E was stable for 3 days up to at 37 degrees C and Hb D and Hb Q for 3 days up to 42 degrees C. This capillary blood collection system would have tremendous potential for sample collection and transportation under adverse climatic conditions for screening of hemoglobinopathies in remote areas in different countries.

HPLC studies in hemoglobinopathies.

An accurate diagnosis of beta -thalassemia carriers, homozygous patients and identification of different structural hemoglobin variants is important for epidemiological studies as well as for management and prevention of the major hemoglobin disorders. There are many electrophoretic and chromatographic approaches for estimation of HbA2 and Hb F but cation exchange HPLC (CE-HPLC)using automated dedicated machines like the Variant Hb testing system have become the method of choice for these investigations. CE-HPLC also helps in the presumptive identification of many abnormal hemoglobin variants and has been useful for both neonatal screening of sickle cell disease as well as second trimester prenatal diagnosis of thalassemia by fetal blood analysis. Other applications of HPLC in hemoglobinopathies include separation of globin chains, measuring the ratio of gamma globin chains (Ggamma/Agamma) and the recently described denaturing HPLC for detecting mutations in both alpha and beta globin genes.

Reduced pancreatic polypeptide response to hypoglycemia and amylin response to arginine in subjects with a mutation in the HNF-4alpha/MODY1 gene.

Subjects with the Q268X mutation in the hepatocyte nuclear factor (HNF)-4alpha gene (RW pedigree/maturity-onset diabetes of the young [MODY]-1) have diminished insulin and glucagon secretory responses to arginine. To determine if pancreatic polypeptide (PP) secretion is likewise involved, we studied PP responses to insulin-induced hypoglycemia in 17 RW pedigree members: 6 nondiabetic mutation-negative [ND(-)], 4 nondiabetic mutation-positive [ND(+)], and 7 diabetic mutation-positive [D(+)]. Subjects received 0.08 U/kg body wt human regular insulin as an intravenous bolus to produce moderate self-limited hypoglycemia. PP areas under the curve (PP-AUCs) were compared among groups. With hypoglycemia, the PP-AUC was lower in the D(+) group (14,907 +/- 6,444 pg/ml, P = 0.03) and the ND(+) group (14,622 +/- 6,015 pg/ml, P = 0.04) compared with the ND(-) group (21,120 +/- 4,158 pg/ml). In addition, to determine if the beta-cell secretory defect in response to arginine involves amylin in addition to insulin secretion, we analyzed samples from 17 previously studied RW pedigree subjects. We compared the AUCs during arginine infusions for the 3 groups both at euglycemia and hyperglycemia as well as their C-peptide-to-amylin ratios. The D(+) and ND(+) groups had decreased amylin AUCs during both arginine infusions compared with the ND(-) group, but had similar C-peptide-to-amylin ratios. These results suggest that the HNF-4alpha mutation in the RW/MODY1 pedigree confers a generalized defect in islet cell function involving PP cells in addition to beta- and alpha-cells, and beta-cell impairment involving proportional deficits in insulin and amylin secretion.

Expression and characterization of HCV NS3 protease.

The overexpression of a gene in a heterologous system is often the prelude to or the prerequisite of the elucidation and characterization of a given protein, in particular where the protein is difficult to obtain in sufficient quantity from natural sources. Prokaryotic expression systems, in particular Escherichia coli have been exploited successfully for a number of viral proteins. A large number of E. coli expression systems are currently commercially available that offer the investigator a large number of choices with regard to promoter choice, site of expression, fusions, and so forth. Since the variety and number of expression systems available are extensive, it is not within the scope of this chapter to discuss them fully, and the final choice of expression system used, is often arrived at empirically and often reflects the investigator's "favored system."

Fas and Fas ligand expression in the salivary glands of patients with primary Sjögren's syndrome.

OBJECTIVE: To assess the role of Fas-mediated apoptosis in the salivary glands of patients with primary Sjögren's syndrome (SS). METHODS: Expression of Fas, Fas ligand (FasL), and bcl-2 in salivary gland biopsy material was detected in situ by immunohistochemical staining and reverse transcriptase-polymerase chain reaction. DNA fragmentation in apoptotic cells was assessed by the enzymatic incorporation of labeled nucleotides (digoxigenin-dUTP). RESULTS: The acinar epithelial cells in SS were Fas+ and FasL+, and these cells died by apoptosis. The majority of infiltrating lymphocytes in SS were Fas+ and bcl-2+, while few lymphocytes expressed FasL. In situ detection of apoptosis showed minimal cell death of lymphocytes, particularly in dense periductal foci. Lymphocytic cell death was significantly lower (P < 0.0001) in these foci compared with that in the interstitium. CONCLUSION: Infiltrating lymphocytes in the focal lesions of the salivary glands of patients with SS are blocked in their ability to commit to apoptosis, even though they may express Fas. The presence of bcl-2 in these cells may explain their inability to undergo apoptosis. The acinar epithelial cells, in contrast, may undergo Fas-mediated apoptosis. These results suggest that the Fas death pathway may be an important mechanism leading to the glandular destruction found in SS.

Primary Sjögren's syndrome in men.

OBJECTIVE: To describe the clinical expression of primary Sjögren's syndrome (SS) in men, focusing on extraglandular manifestations (EGM) and serological markers of disease. METHODS: In a cross sectional and comparative study, adult men with primary SS were identified from a cohort study on SS, and 26 age matched adult women with primary SS were selected as a control group. All patients met the European classification criteria for SS. They were compared for demographic, clinical and laboratory findings. RESULTS: Thirteen men with primary SS were identified. Mean age at onset was 39 (SEM 4) years and mean duration of disease was 7.8 (1) years. Sicca complex or parotitis was the presenting feature in eight patients (61.5%), and an EGM in five (38.5%). During the course of the disease, EGM were present in 12 patients (92%), polyarthralgias and lymphopenia being the most frequent (38.5% each). Rheumatoid factor was positive in 73% of patients, antinuclear antibodies in 85%, anti-(SS-A) in 62%, and anti-(SS-B) in 46%. No statistical differences in the frequency of EGM or in the presence of autoantibodies were observed between men and women. However, men patients were more likely to have EGM. CONCLUSION: Primary SS in men is an uncommon condition with clinical and serological characteristics similar to those observed in women. Sex hormones may be incriminated in the pathogenesis of SS. However, it remains poorly understood whether sex hormones play a major role in the severity of disease and have any importance with regard to treatment.

In vitro cleavage of hepatitis C virus polyprotein substrates by purified recombinant NS3 protease.

The non-structural protein NS3 of hepatitis C virus has been expressed in bacteria as a polyhistidine fusion protein which can be produced in a soluble form and easily purified by affinity chromatography. Using an in vitro transcription and translation system we have been able to demonstrate that this protein can proteolytically process substrate molecules derived from the non-structural region of the polyprotein. Using this assay system we have been able to optimize basic biochemical characteristics of the purified enzyme. Parallel experiments show that the full-length NS3 protein also possesses ATPase activity, indicating the bifunctional nature of the protein. In contrast, purified NS3 in which the predicted catalytic serine has been mutated loses protease but retains ATPase activity.

Analysis of NS3-mediated processing of the hepatitis C virus non-structural region in vitro.

The protease activity of the hepatitis C virus (HCV) NS3 protein has been investigated using transient expression methods in mammalian cells, as well as in vitro transcription/translation systems. We confirmed that expression of the NS3-5 polyprotein in rabbit reticulocyte lysates results in efficient cis processing at the NS3/NS4 junction. However, processing at the other predicted sites of NS3-mediated cleavage varied markedly in efficiency, the site most susceptible being that between NS5A and NS5B. Time-course analysis of the proteolytic processing of the HCV non-structural precursor showed that the cis cleavage between NS3 and NS4 occurred extremely rapidly. However, efficient cleavage at this position was dependent on the prior removal of the NS2 protein. Furthermore, the presence of uncleaved NS2 sequences on the enzyme severely impeded NS3-mediated proteolysis at downstream sites in the polyprotein. This suggests therefore that efficient cleavage at the NS2/NS3 junction is a pivotal event in HCV replication. During the course of this study a proteolytically inactive mutant of NS3 was characterized carrying a previously unreported amino acid substitution near the proposed active site of the enzyme. Molecular modelling suggested that the amino acid present at this position may influence the conformation of the active site of the enzyme. Recently a number of reports have described a second protease activity, located in the NS2/NS3 region, which is responsible for cleavage at the NS2/NS3 junction. We have identified an isolate of HCV, obtained from a U.K. patient, which has a virtually inactive NS2/NS3 protease. The possible implications of this observation are discussed.

Two-step binding mechanism for HIV protease inhibitors.

Rate constants for binding of five inhibitors of human immunodeficiency virus (HIV) protease were determined by stopped-flow spectrofluorometry. The two isomers of quinoline-2-carbonyl-Asn-Phe psi-[CH(OH)CH2N]Pro-O-t-Bu (R diastereomer = 1R; S diastereomer = 1S) quenched the protein fluorescence of HIV protease and thus provided a spectrofluorometric method to determine their binding rate constants. The dissociation rate constants for acetyl-Thr-Ile-Leu psi(CH2NH)Leu-Gln-Arg-NH2 (2), (carbobenzyloxy)-Phe psi[CH(OH)CH2N]Pro-O-t-Bu (3), and pepstatin were determined by trapping free enzyme with 1R as 2, 3, and pepstatin dissociated from the respective enzyme.inhibitor complex. Association rate constants of 1R, 2, and pepstatin were calculated from the time-dependent inhibition of protease-catalyzed hydrolysis of the fluorescent substrate (2-aminobenzoyl)-Thr-Ile-Nle-Phe(NO2)-Gln-Arg-NH2 (4). The kinetic data for binding of 1S to the protease fit a two-step mechanism. Kd values for these inhibitors were calculated from the rate constants for binding and were similar to the respective steady-state Ki values.

A cassette vector for the construction of antigen chimaeras of poliovirus.

A cassette vector has been constructed which allows the rapid and extensive modification of one of the neutralizing antigenic sites of the Sabin 1 poliovirus vaccine strain, P1/LSc 2ab. Unique restriction endonuclease sites flanking antigenic site 1 have been engineered into a full-length infectious Sabin 1 cDNA clone with minimal alteration to the coding sequence. This facilitates replacement of this region by oligonucleotides encoding foreign amino acid sequences. Our results indicate that this region is highly flexible in terms of the number and sequence of amino acids which can be accommodated.