Evaluation of neonatal screening for congenital adrenal hyperplasia

Neonatal screening for congenital hypothyroidism has been effective in early detection and treatment of the condition. The position with respect to neonatal screening for congenital adrenal hyperplasia has been debated for many years. Some countries have performed congenital adrenal hyperplasia screening for many years, others have conducted pilot studies that were then not adopted. This article endeavours to summarize the complex issues behind decisions whether to screen or not and summarizes the findings of neonatal congenital adrenal hyperplasia screening programmes.     

Rapid steroid hormone quantification for congenital adrenal hyperplasia (CAH) in dried blood spots using UPLC liquid chromatography-tandem mass spectrometry

Newborn screening for congenital adrenal hyperplasia (CAH) is usually done by quantifying 17α-hydroxyprogesterone using immunoassay. However, this test produces high rates of false positive results caused by cross reacting steroids. Therefore we have developed a selective and specific method with a short run time (1.25 min) for quantification of 17α-hydroxyprogesterone, 21-deoxycortisol, 11-deoxycortisol, 11-deoxycorticosterone and cortisol from dried blood spots. The extraction procedure is very simple and steroid separation is ensured on a BEH C18 column and an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Analysis was done in positive ionization mode (ESI+) and recorded in multiple reaction monitoring mode (MRM). The method gave linear results for all steroids over a range of 5-200 (cortisol: 12.5-500) nmol/L with coefficients of regression >0.992. Absolute recovery was >64.1%. Across the analytical range the inter-assay coefficient of variation (CV) was <3%. Newborn blood samples of patients with confirmed 21-CAH and 11-CAH could clearly be distinguished from samples of unaffected newborns falsely positive on immunoassay. The method is not influenced by cross reactions as found on immunoassay. Analysis of dried blood spots shows that this method is sensitive and fast enough to allow rapid analysis and can therefore improve the newborn screening program.     

Neonatal screening for congenital adrenal hyperplasia in North-Eastern Italy: a report three years into the program

AIMS: To evaluate the incidence of congenital adrenal hyperplasia (CAH) in the Northern Italian population and the efficiency of the North-Eastern Italy screening program. To adjust cut-off levels for 17-hydroxyprogesterone (17-OHP) in relation to gestational age and birth weight, comparing the benefits in terms of reduction of recall rates with the two approaches and ultimately choosing the better of the two. SUBJECTS AND METHODS: Since September 2001, blood samples from neonates born in North-Eastern Italy have been screened with a fluoroimmunoassay method for 17-OHP determination (DELFIA). A preliminary cut-off level of > or = 30 nmol/l was set both for term and preterm newborns. The values of 17-OHP were analysed using statistical methods in relation to gestational age and birth weight in order to modify the cut-off on the basis of our data. RESULTS: After 33 months of screening we screened 128,282 newborns and detected 6 affected babies. During the first 8 months of screening among the recalled babies, 89.6 and 78.1% were preterm and low-birth-weight newborns, respectively, with a recall rate of 2.59% for premature neonates and of 4.94% for babies with birth weights < 2,500 g. We chose a new cut-off value of 50 nmol/l for preterm newborns only and, after 4 months, the recall rate was reduced to 0.83% for these infants and to 1.83% for low-birth-weight infants. CONCLUSION: After 33 months of screening for CAH in North-Eastern Italy, we report an incidence of 1:21,380. In 5 out of 6 affected babies, the diagnosis was established only after a positive screening test, which prevented a severe salt-wasting crisis in these babies. The cut-off level related to gestational age led to a significant reduction in the number of false-positives among preterm babies.We therefore intend to continue with the screening program for CAH in North-Eastern Italy, keeping a gestational-age-related cut-off in the hope that our data may encourage a national screening program for CAH.     

DNA microextraction from dried blood spots on filter paper blotters: potential applications to newborn screening

Summary Microextraction of DNA from dried blood specimens would ease specimen transport to centralized laboratory facilities for recombinant DNA diagnosis in the same manner as use of dried blood spots allowed the broad application of screening tests to newborn populations. A method is described which reproducibly yields 0.5g DNA from the dried equivalent of 50l whole blood. Though DNA yields decreased with storage of dried specimens at room temperature, good-quality DNA was still obtained. Sufficient DNA was routinely obtained for Southern blot analysis using repetitive and unique sequences. This microextraction procedure will allow immediate application of molecular genetic technology to direct newborn screening follow-up of disorders amenable to DNA diagnosis, such as sickle cell anemia, and may eventually permit primary DNA screening for specific mutations.     

17OH-progesterone response to acute dexamethasone administration in congenital adrenal hyperplasia

Glucocorticoid-related changes in 17OH-progesterone (17P) concentrations were studied in 13 patients receiving treatment for 21-hydroxylase deficiency congenital adrenal hyperplasia (CAH). Blood spot and saliva 17P levels were elevated, with or without loss of diurnal rhythm, within 24 h of stopping maintenance glucocorticoid therapy. A single dose of dexamethasone (0.01 mg/kg) given either intravenously or orally at 09.00 h resulted in rapid onset of complete pituitary-adrenal suppression characterised by a prompt and exponential fall in 17P levels (first-order elimination half-life, mean 2.87 h, range 1.98-3.98 h). Concentrations of 17P remained low throughout the day until the onset of an abrupt nocturnal rise, which occurred between 24.00 and 05.00 h. There were individual differences in both the rate of fall and the timing of the nocturnal rise in 17P levels which may partly explain the need to vary individual steroid requirements in the treatment of CAH.     

Procedure for neonatal screening for congenital adrenal hyperplasia due to 21-hydroxylase deficiency

The value of screening of neonates for congenital adrenal hyperplasia is not universally accepted. Procedures for screening are recommended here in order to provide a structure to the testing and ultimately bring together data that will allow the effect of screening to be judged for benefit or dismissed as no better than clinical recognition of the disease state.     

Mineralocorticoids in congenital adrenal hyperplasia

While hypertension is observed in only two of the three major subtypes of congenital adrenal hyperplasia (CAH), 11β- and 17-hydroxylase deficiencies, deoxycorticosterone ( (DOC) production is increased in all. The elevated zona fasciculata (ZF) DOC produces mineralocorticoid hypertension with suppressed renin and reduced potassium concentrations. The DOC levels in 21-hydroxylase deficiency are in part produced by renin stimulation of the Zona glomerulosa (ZG) along with aldosterone. Assessment of the mineralocorticoid hormones of the ZF and ZF (17-deoxy steroids) provides additional unique characteristics of each subtype. Dissociation of DOC from cortisol is not unique to CAH. This dissociation is seen in other disorders and contrived conditions. There is a strong suggestion of a non-ACTH regulator of 17-deoxy steroids (DOC) that may contribute significantly to DOC production in general and effect DOC levels in CAH.     

Control in congenital adrenal hyperplasia monitored by frequent saliva 17OH-progesterone measurements

Treatment in a group of 19 patients with congenital adrenal hyperplasia (CAH) has been monitored by frequent, serial measurements of saliva 17OH-progesterone (17OHP) concentrations. Detailed 17OHP profiles were obtained during consecutive weekend days and every 1-2 h over a separate 24-hour period. Patients showed a marked diurnal rhythm in 17OHP levels, particularly when treated with hydrocortisone. In some patients, 10 mg/m2/day of hydrocortisone was sufficient glucocorticoid replacement to produce adequate control, although there was considerable individual variation. Saliva 17OHP profiles provided valuable information on the efficacy of hydrocortisone, cortisone acetate, prednisolone and dexamethasone as glucocorticoid suppressive regimes in the treatment of CAH. Preliminary results suggest that hydrocortisone given in two divided doses during the day, supplemented by a small dose of prednisolone at bedtime, is suitable treatment for CAH patients who are still growing. In the patient who has completed statural growth, single daily dose dexamethasone therapy ensures adequate adrenal suppression and is convenient in the longterm.     

A paper membrane filter assay for ciliate chemoattraction

A quantitative bioassay for ciliate chemoattraction based on the Boyden assay is described with the ciliates Tetrahymena thermophila and Tetrahymena pyriformis as test organisms. A chamber is separated into two compartments by a Whatman 3MM filter, and a suspension of starved cells (approximately 10(5) cells/ml) is placed in one compartment and a solution containing attractant in the other. The gradient of chemoattractant across the filter causes the cells to swim through the filter into the attractant-containing compartment where their appearance is determined by electronic cell counting. The assay described is convenient with a signal-to-noise ratio of approximately 10. It is shown here to work with the attractants proteose peptone and platelet-derived growth factor.     

Using dried blood spots stored on filter paper to measure cholinesterase activity in wild avian species

Birds of prey that are poisoned by cholinesterase inhibitors (e.g. organophosphate and carbamate insecticides) are often cared for at animal shelters, rehabilitation centres and wildlife diagnostic facilities. Plasma cholinesterase (ChE) activity is a recognized method of assessing exposure to these insecticides, but standard blood-handling protocols are difficult to follow in non-laboratory settings. The primary objective of this study was to expand upon a method for storing human blood on filter paper without the need for complicated equipment or refrigeration, and to test its utility for measurement of ChE activity in avian blood. ChE activity from whole blood, plasma, and dried blood spots was analysed from 169 wild birds and comparisons made among sample types. ChE activity measured in whole blood haemolysates and dried blood spots were significantly correlated (r=0.74, p<0.001), as was ChE activity measured in plasma and dried blood spots (r=0.68, p<0.001). This study demonstrated that monitoring pesticide exposure in birds could be conducted using elementary blood sampling, preserving and shipping techniques.     

Female preponderance in congenital adrenal hyperplasia due to CYP21 deficiency in England: implications for neonatal screening

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase (CYP21) deficiency causes symptoms ranging from life-threatening neonatal adrenal crises to minimal virilization in adulthood. The relationship between CYP21 genotype and phenotypic markers in a non-screened population of 73 CAH children (44 female, 29 male; 54 white, 19 Asian) treated at the Royal Manchester Children's Hospital was investigated and ethnic and sex differences assessed. The patients were categorized according to the mutation on the mildest allele. The age at the time of diagnosis differed significantly between the groups (p = 0.02): all 25 Null and 25 of 26 of the I2 splice patients were diagnosed during the neonatal period, whereas 7 of 11 I172N patients were diagnosed late. Degree of female genital virilization, 17-hydroxyprogesterone level at diagnosis, and fludrocortisone requirement during the 1st year of treatment correlated with the genotype, although Asian Null patients required more fludrocortisone than their white counterparts (p = 0.055). There was an equal sex ratio in both the I2 splice (12 female/14 male) and I172N (5 female/6 male) groups. However, in the Null group, the ratio was 4.0 (20 female/5 male; p = 0.003), suggesting that some Null male infants perish before being clinically detected to have CYP21 deficiency. Our findings strongly support the need for implementation of a neonatal screening programme for CAH in the UK which may reduce the male infant mortality.     

Serum 17-hydroxypregnenolone and 17-hydroxypregnenolone sulfate concentrations in patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency

Serum concentrations of 17-hydroxypregnenolone, 17-hydroxypregnenolone sulfate and 17-hydroxyprogesterone were measured simultaneously in patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency, using a combined radioimmunoassay method. All these precursor steroids were found to be markedly elevated in the sera of untreated patients with a salt-losing form of the disease, whereas, in untreated patients with a simple virilizing form, only the concentration of unconjugated steroids was increased and the 17-hydroxypregnenolone sulfate concentration remained within the normal range. Among the patients with a salt-losing form under maintenance therapy, these steroids were all still significantly increased in those on insufficient control, whereas only 17-hydroxyprogesterone was significantly but slightly increased in those on adequate control. Although the mechanism whereby the serum 17-hydroxypregnenolone sulfate concentration is not increased in the untreated simple virilizers is unknown, both a milder degree of 21-hydroxylase deficiency and a role of 17-hydroxypregnenolone sulfate in adrenal steroid production as a kind of supplier are suggested as possible explanations, especially in the neonatal period and early infancy. Thus, this study showed the serum concentrations of 17-hydroxypregnenolone and its sulfate together with 17-hydroxyprogesterone in patients with 21-hydroxylase deficiency in various conditions.     

Value of 17-hydroxyprogesterone determination in exploration of adrenal cortex enzyme deficiencies]

17-hydroxyprogesterone (17-OH-P) was measured in various populations by radioimmunoassay, using a highly specific antibody produce in the rabbit. Dynamic tests were performed with ACTH, dexamethasone and estroprogestative drugs and the role played by the adrenals and the ovaries in 17-OH-P production could be assessed. 17-OH-P determination is of interest in that, it allows the diagnosis of 21-hydroxylase deficiency, where values above 10 ng/ml are often found. Associated with the measure of testosterone and delta 4-androstenedione, it is also useful in the management of the disease. In the mild form of congenital adrenal hyperplasia with late revelation of the symptoms, determination of 17-OH-P following ACTH stimulation allows of relative diagnosis.     

Genetic linkage studies between congenital adrenal hyperplasia and the HLA blood group system

Seventeen families with one or two children suffering from congenital adrenal hyperplasia (CAH) were not only typed for their HLA-A,B, and D antigens but also tested biochemically forCAH heterozygosity after ACTH stimulation. The lod score analysis showed a close genetic linkage betweenCAH andHLA, indicating that theC-21-hydroxylase deficiency gene(s) causing CAH in the homozygous deficient state are-located in close proximity to theHLA complex on chromosome 6 with an estimated recombination fraction of 0 to 5%. HLA typing in 21 unrelated CAH patients revealed a statistically significant association to the HLA-B5 antigen with a relative risk value of 5.8. There was a significant correlation (P=0.0025) between theHLA segregation data and theCAH heterozygosity test results in relatives of CAH patients although a few ‘false negative’ results in theCAH heterozygosity test were observed. Thus, the combination of HLA typing and this biochemical test at present provides the most precise approach for detecting CAH carriers in families of CAH patients.