Svetlana Lajic

Long-term follow-up of prenatally treated children at risk for congenital adrenal hyperplasia : Does dexamethasone cause behavioural problems?

OBJECTIVES To investigate the long-term effects of prenatal treatment of congenital adrenal hyperplasia (CAH) with emphasis on behavioural problems and temperament. DESIGN A population-based long-term follow-up study of Swedish children at risk for virilising CAH, who had received treatment prenatally with dexamethasone (DEX). The questionnaire-based follow-up was performed when the children had reached school age. METHODS Standardised parent-completed questionnaires were used to evaluate adaptive functioning, behavioural/emotional problems and psychopathology, social anxiety and temperament in DEX-exposed school-aged children (n=26) and matched controls (n=35). In addition, the association between parental questionnaires and childrens self-ratings was investigated. RESULTS There were no statistically significant differences between DEX-exposed children and controls in measures of psychopathology, behavioural problems and adaptive functioning. In a questionnaire on temperamental traits, DEX-exposed children were described by their parents as being more sociable than controls (P=0.042). The correlation analysis showed only modest parent-child agreement on social anxiety, i.e. the increased social anxiety in childrens self-ratings was not confirmed by their parents. CONCLUSIONS DEX-treated children showed good overall adjustment. The parent-child agreement with respect to social anxiety was modest, highlighting the importance of multiple information sources and assessment methods. The clinical significance of the observed difference in sociability cannot be determined within the frameworks of this study. Additional studies of larger cohorts are essential to make more decisive conclusions on the safety of the treatment. Until then, it is important that parents are thoroughly informed about the benefits and potential risks and uncertainties of this controversial treatment.

High self-perceived stress and many stressors, but normal diurnal cortisol rhythm, in adults with ADHD (attention-deficit/hyperactivity disorder).

Attention-deficit/hyperactivity disorder (ADHD) in adults is associated with significant impairment in many life activities and may thus increase the risk of chronic stress in everyday life. We compared adults with a DSM-IV ADHD diagnosis (n=28) with healthy controls (n=28) regarding subjective stress and amounts of stressors in everyday life, diurnal salivary cortisol in the everyday environment and salivary cortisol before and after cognitive stress in a laboratory setting. The association between cortisol concentrations and impulsivity was also investigated. Consistent with assumptions, individuals with ADHD reported significantly more self-perceived stress than controls, and subjective stress correlated with the amount of stressors in everyday life. The two groups were comparable with respect to overall diurnal cortisol levels and rhythm, as well as in pre- and post-stress cortisol concentrations. Post-stress cortisol (but not baseline cortisol) concentration was positively correlated with impulsivity. The group with high post-stress cortisol also reported more symptoms of depression and anxiety, as well as self-perceived stress and stressors in every-day life. The diagnosis of ADHD significantly increased the risk of belonging to the group with high post-stress cortisol levels. The results in this study warrant a focus not only on the primary diagnosis of ADHD, but also calls for a broader assessment of stressors and subjective stress in everyday life, as well as support comprising stress management and coping skills.

Long-term outcome of prenatal treatment of congenital adrenal hyperplasia.

Prenatal treatment of congenital adrenal hyperplasia (CAH) with dexamethasone to minimize the genital virilization of external genitalia of affected girls has been in use since the mid-1980s. The positive effect of reducing virilization is now established. However, experimental data from animal studies and observations on adverse medical events in human newborns have raised concerns about the long-term safety of the treatment. Most animal studies on prenatal treatment with synthetic glucocorticoids have been designed to mimic treatment for lung maturation in preterm infants. The primary focus has been on a possible impact on fetal programming and the development of the metabolic syndrome with insulin resistance, type 2 diabetes, and high blood pressure. Altered reactivity to stress as a function of differences in reactivity of the HPA axis and glucocorticoid receptor function have been assayed. Effects on cognition, especially memory, have been observed. In children at risk for CAH and treated prenatally with dexamethasone, no overall effects on full-scale IQ have been observed, but a negative effect on verbal working memory has been reported. Contradictory effects on social behavior with respect to shyness and inhibition have been discussed. There is an urgent need for in-depth studies of long-term outcome in prenatal treatment of CAH regarding both maternal side effects and possible negative metabolic as well as cognitive and behavioral effects in the growing fetus and the child in her development into adulthood.

A cluster of missense mutations at Arg356 of human steroid 21-hydroxylase may impair redox partner interaction

Abstract Lesions in the gene encoding steroid 21-hydroxylase result in congenital adrenal hyperplasia, with impaired secretion of cortisol and aldosterone from the adrenal cortex and overproduction of androgens. A limited number of mutations account for the majority of mutated alleles, but additional rare mutations are responsible for the symptoms in some patients. A total of 11 missense mutations has previously been implicated in this enzyme deficiency. We describe two novel missense mutations, both affecting the same amino acid residue, Arg356. The two mutations, R356P and R356Q, were reconstructed by in vitro site-directed mutagenesis, the proteins were transiently expressed in COS-1 cells, and enzyme activity towards the two natural substrates, 17-hydroxyprogesterone and progesterone, was determined. The R356P mutant reduced enzyme activity to 0.15% towards both substrates, whereas the R356Q mutant exhibited 0.65% of normal activity towards 17-hydroxyprogesterone, and 1.1% of normal activity towards progesterone. These activities correspond to the degrees of disease manifestation of the patients in whom they were found. Arg356 is located in a region which recently has been implicated in redox partner interaction, by modelling the structure of two other members of the cytochrome P450 superfamily. Of the 11 previously described missense mutations, three affect arginine residues within this protein domain. With the addition of R356P and R356Q, there is a clear clustering of five mutations to three closely located basic amino acids. This supports the model in which this protein domain is involved in redox partner interaction, which takes places through electrostatic interactions between charged amino acid residues.

Inhibition of CYP21A2 Enzyme Activity Caused by Novel Missense Mutations Identified in Brazilian and Scandinavian Patients

BACKGROUND Most patients with 21-hydroxylase deficiency carry CYP21A1P-derived mutations, but an increasing number of novel and rare mutations have been reported in disease-causing alleles. OBJECTIVE Functional effects of three novel (p.G56R, p.L107R, p.L142P) and one recurrent (p.R408C) CYP21A2 mutations were investigated. The degree of enzyme impairment caused by p.H62L alone or combined to p.P453S was also analyzed. DESIGN The study included 10 Brazilian and two Scandinavian patients. To determine the deleterious role of each mutant protein, in vitro assays were performed in transiently transfected COS-1 cells. For a correct genotype-phenotype correlation, the enzymatic activities were evaluated toward the two natural substrates, 17-hydroxyprogesterone and progesterone. RESULTS Low levels of residual activities obtained for p.G56R, p.L107R, p.L142P, and p.R408C mutants classified them as classical congenital adrenal hyperplasia mutations, whereas the p.H62L showed an activity within the range of nonclassical mutations. Apparent kinetic constants for p.H62L confirmed the nonclassical classification as the substrate binding capacity was within the same magnitude for mutant and normal enzymes. A synergistic effect was observed for the allele bearing the p.H62L+p.P453S combination because it caused a significant reduction in the enzymatic activity. CONCLUSIONS We describe the functional analysis of five rare missense mutations identified in Brazilian and Scandinavian patients. The p.G56R, p.L107R, and p.L142P are reported for the first time. Most probably these novel mutations are closer to null than the p.I172N, but for the p.G56R, that might not be the case, and the p.H62L is definitely a nonclassical mutation.

Naturally Occurring Mutants of Human Steroid 21-Hydroxylase (P450c21) Pinpoint Residues Important for Enzyme Activity and Stability

Three mutants (deletion of E196, G291S, and R483P) of steroid 21-hydroxylase (P450c21) from patients with inherited congenital adrenal hyperplasia had reduced activity toward progesterone and 17-hydroxyprogesterone after transient expression in cultured mammalian cells. In addition, both the E196 deletion and the R483P mutant had shorter half-lives than the wild-type enzyme, whereas the half-life of the G291S mutant was comparable with that of the normal protein. These results directly link the clinical situation with the three mutations and suggest that G291 is important for the catalytic activity of P450c21.

Prenatal Dexamethasone Treatment of Children at Risk for Congenital Adrenal Hyperplasia: The Swedish Experience and Standpoint

Congenital adrenal hyperplasia (CAH) refers to rare ( 1:10,000), recessively inherited disorders of adrenal steroidogenesis. 21-Hydroxylase deficiency, the most common form, results in virilization of external genitalia in severely affected female fetuses. Prenatal treatment of CAH with dexamethasone (DEX) has been administered since the mid-1980s and is effective in reducing virilization in CAH-affected girls (1). The treatment has to be initiated early in pregnancy, before prenatal testing is possible. Consequently, seven of eight fetuses (all boys and CAH-unaffected girls) are treated in early pregnancy without any benefit of the treatment per se, until prenatal testing can determine the sex and diagnostic status at the end of the first trimester. CAH-affected girls are treated until term. Prenatal DEX treatment of fetuses at risk for CAH is controversial. Results from animal studies, as well as studies on prenatal glucocorticoid therapy in other contexts, have raised concerns regarding possible negative effects on behavioral and somatic development (2, 3). Adult corticosteroid-exposed rodent offspring are characterized by hypertension (via reduced nephron number); hyperinsulinemia and hyperglycemia (via permanently increased levels of the hepatic enzyme phosphoenolpyruvate carboxylase); fatty liver but not obesity when given a high-fat diet; and hyperactivity of the hypothalamic-pituitaryadrenal axis and altered affective behavior, reminiscent of anxiety, as well as impaired learning and memory functions. In the rhesus monkey, prenatal DEX during the last trimester resulted in offspring with elevated basaland stress-stimulated cortisol at the age of 10 months as well as smaller hippocampal volume. In the African vervet monkey, high-dose DEX exposure (120–200 g/kg) from midgestation until term resulted in offspring with an exaggerated cortisol response to stress already prepubertally, and adult animals exhibited hypertension, impaired glucose tolerance, and hyperinsulinemia (4). A long-term follow-up of individuals treated prenatally with betamethasone due to a risk of preterm birth shows that 30 yr after exposure the individuals exhibit insulin resistance, particularly in women, and 7% of the adults had elevated basal, morning cortisol levels (5). Other authors consider the prenatal treatment of CAH to be safe (1), but the recent Endocrine Society Guidelines state that such treatment should only be done within the frames of ethically approved clinical studies (6). The prenatal treatment of CAH is considered to be safe in the short-term perspective, although there are few longterm follow-up studies. In Sweden, prenatal treatment of CAH has been administered within the frameworks of a clinical study since 1999. The study, which is ongoing, is designed as a prospective, nonrandomized, multicenter trial in several participating European countries, including both follow-up of the mothers and long-term follow-up of the children. In Sweden, 31 cases have been enrolled during the last 10 yr. Hence, it will take several years before the first results from the long-term follow-up assessments will be available. We have also conducted retrospective follow-up studies of mothers and 43 children treated in Sweden and Nor-

An intron 1 splice mutation and a nonsense mutation (W23X) in CYP21 causing severe congenital adrenal hyperplasia

Abstract Direct DNA sequencing of the steroid 21-hydroxylase gene (CYP21) revealed two novel mutations in two patients with severe congenital adrenal hyperplasia. The nonsense mutation Trp23Stop (TGG → TGA) was found in a woman with the simple virilizing form of the disease. She was a compound heterozygote, with the previously described Ile173Asn mutation on her other allele. A boy, who developed salt-wasting in the neonatal period, carried an allele with a novel mutation of the canonical splice acceptor site in intron 1 (AG→GG). He was also a compound heterozygote, with the well-known splice mutation in intron 2 on his other allele.

Gender role behaviour in prenatally dexamethasone-treated children at risk for congenital adrenal hyperplasia--a pilot study.

Aim:  To investigate the effects of prenatal dexamethasone (DEX) exposure on gender role behaviour.

Functional studies of two novel and two rare mutations in the 21-hydroxylase gene

Congenital adrenal hyperplasia (CAH) is most commonly due to 21-hydroxylase deficiency and presents with a wide spectrum of clinical manifestations, from prenatal virilization and salt-wasting in the neonatal period to precocious pubarche and late-onset hyperandrogenic symptoms during adulthood. A limited number of mutations account for the majority of all mutated alleles, but a growing number of rare mutations are responsible for the disease in some patients. By sequence analysis of the CYP21A2 gene, we identified two novel (I171N and L446P) and two rare (R341P and R426H) mutations in seven Italian patients with CAH. One of the patients was diagnosed with mild non-classical CAH and was found to be a compound heterozygote (I171N/V281L), while all other patients showed severe phenotypes with latent or manifest salt-wasting. The residual activities measured after expression of the four mutant enzymes in COS-1 cells were all below 1% towards both natural substrates (17-OH-progesterone and progesterone) compared with the wild-type protein. All four mutations are, thus, associated with severe enzyme deficiency and are predicted to cause classic CAH if found in trans with other mutations causing severe enzyme deficiency.