ALIS‐FLP: Amplified ligation selected fragment‐length polymorphism method for microbial genotyping

A DNA fingerprinting method known as ALIS‐FLP (amplified ligation selected fragment‐length polymorphism) has been developed for selective and specific amplification of restriction fragments from TspRI restriction endonuclease digested genomic DNA. The method is similar to AFLP, but differs in that only one specific restriction enzyme (TspRI) is used. The cohesive ends of the DNA fragments are ligated with two types of oligonucleotide. A long oligonucleotide containing the primer site and the specific 9 nt 3 prime end, which is complementary to specific 9 nt, cohesive 3 prime end of the TspRI genomic DNA fragment, and a short, degenerated, oligonucleotide covering the remaining TspRI cohesive ends. Other cohesive ends are covered by a short degenerated oligonucleotide lacking the primer site. The ligation mixture is used as a template for amplification using a single primer corresponding to the 5 prime end of the long, specific oligonucleotide. The selection of TspRI digested genomic DNA fragments for amplification is achieved by sequence selective ligation of the specific long oligonucleotide carrying the primer site to both ends of the specific target fragment. This technique allows for differentiation of the organisms without previous knowledge of their DNA sequence. The usefulness of the method is confirmed by genotyping of 70 previously characterized clinical E. coli isolates. The grouping obtained was identical to the results of REA‐PFGE. Versatility of the method is highlighted, i.e. its combining the advantages of the AFLP technique with a simple, rapid and cheap polymerase chain reaction product detection method.     

Estimation of the false-negative rate in newborn screening for congenital adrenal hyperplasia

OBJECTIVE: Newborn screening based on measurement of 17alpha-hydroxyprogesterone (17-OHP) in a dried blood spot on filter paper is an effective tool for early diagnosis of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. Its most important rationale is prevention of a life-threatening salt-wasting (SW) crisis; in moderate forms of CAH, early diagnosis and treatment may prevent permanent negative effects of androgen overproduction. Our target was to analyse if all CAH patients who had been identified clinically before puberty would have been detected by the newborn screening. METHODS: Newborn screening cards of 110 CAH patients born between 1988 and 2000 in five Middle-European countries and diagnosed prior to puberty (77 SW and 33 moderate) and cards from 920 random, healthy newborn controls were analysed. CAH screening had not yet been introduced during this time. The diagnosis was based on clinical and laboratory signs and, in most cases, on CYP21 gene mutation analysis. All 17-OHP measurements in dried blood spots were carried out using a time-resolved fluoroimmunoassay kit. RESULTS: In the newborn screening blood spots, the median of 17-OHP levels was 561 nmol/l (range 91-1404 nmol/l) in subjects with the SW form and 40 nmol/l (4-247 nmol/l) in the moderate form. All 77 SW patients would have been detected by newborn screening using the recommended cut-off limits (30 nmol/l). However, 10 of 33 patients with moderate CAH would have been missed. 17-OHP levels of all controls were below the cut-off. CONCLUSION: Newborn screening is efficient for diagnosing the SW form of CAH, but is inappropriate for identifying all patients with a moderate form of CAH. It appears that the false-negative rate is at least one-third in children with the moderate form of CAH.     

Inhibition of soluble epoxide hydrolase counteracts the development of renal dysfunction and progression of congestive heart failure in Ren‐2 transgenic hypertensive rats with aorto‐caval fistula

The detailed mechanisms determining the course of congestive heart failure (CHF) in hypertensive subjects with associated renal dysfunction remain unclear. In Ren‐2 transgenic rats (TGR), a model of angiotensin II (ANG II)‐dependent hypertension, CHF was induced by volume overload achieved by creation of the aorto‐caval fistula (ACF). In these rats we investigated the putative pathophysiological contribution of epoxyeicosatrienoic acids (EETs) and compared it with the role of the renin‐angiotensin system (RAS). We found that untreated ACF TGR exhibited marked intrarenal and myocardial deficiency of EETs and impairment of renal function. Chronic treatment of these rats with cis‐4‐[4‐(3‐adamantan‐1‐yl‐ureido)cyclohexyloxy]benzoic acid (c‐AUCB, 3 mg/L in drinking water), an inhibitor of soluble epoxide hydrolase (sEH) which normally degrades EETs, increased intrarenal and myocardial EETs, markedly improved survival rate, and increased renal blood flow, glomerular filtration rate and fractional sodium excretion, without altering RAS activity. Chronic angiotensin‐converting enzyme inhibition (ACEi) with trandolapril, (6 mg/L in drinking water) improved survival rate even more, and also inhibited the development of renal dysfunction; these beneficial actions were associated with significant suppression of the vasoconstrictor/sodium retaining axis and further activation of the vasodilatory/natriuretic axis of the systemic and intrarenal RAS, without modifying tissue availability of biologically active fatty acid epoxides. In conclusion, these findings strongly suggest that chronic sEH inhibition and chronic treatment with ACEi, each of them altering a different vasoactive system, delay or even prevent the onset of decompensation of CHF in ACF TGR, probably by preventing the development of renal dysfunction.     

Non-invasive prenatal testing for single gene disorders: exploring the ethics

Non-invasive prenatal testing for single gene disorders is now clearly on the horizon. This new technology offers obvious clinical benefits such as safe testing early in pregnancy. Before widespread implementation, it is important to consider the possible ethical implications. Four hypothetical scenarios are presented that highlight how ethical ideals of respect for autonomy, privacy and fairness may come into play when offering non-invasive prenatal testing for single gene disorders. The first scenario illustrates the moral case for using these tests for ‘information only'', identifying a potential conflict between larger numbers of women seeking the benefits of the test and the wider social impact of funding tests that do not offer immediate clinical benefit. The second scenario shows how the simplicity and safety of non-invasive prenatal testing could lead to more autonomous decision-making and, conversely, how this could also lead to increased pressure on women to take up testing. In the third scenario we show how, unless strong safeguards are put in place, offering non-invasive prenatal testing could be subject to routinisation with informed consent undermined and that woman who are newly diagnosed as carriers may be particularly vulnerable. The final scenario introduces the possibility of a conflict of the moral rights of a woman and her partner through testing for single gene disorders. This analysis informs our understanding of the potential impacts of non-invasive prenatal testing for single gene disorders on clinical practice and has implications for future policy and guidelines for prenatal care.