Phyllis W. Speiser

Molecular genetic analysis of nonclassic steroid 21-hydroxylase deficiency associated with HLA-B14,DR1.

Nonclassic steroid 21-hydroxylase deficiency is a frequent, relatively mild disorder of cortisol biosynthesis, characterized by variable signs of postnatal androgen excess. It is inherited as an allelic variant of the gene designated CYP21B, which encodes 21-hydroxylase. CYP21B is located in the HLA histocompatibility complex, and a nonclassic allelic variant is often associated with characteristic HLA antigens--B14,DR1. We cloned and analyzed the CYP21B gene from a patient homozygous for HLA-B14,DR1 who had nonclassic 21-hydroxylase deficiency. Five deviations from the normal genetic sequence of CYP21B were found, but only one appeared likely to affect the functional integrity of the protein: codon 281, GTG, encoding valine, was changed to TTG, leucine. We constructed an oligonucleotide probe corresponding to the mutant DNA sequence surrounding codon 281 and hybridized the probe with DNA samples digested with the restriction endonuclease Taql. Samples from eight patients with nonclassic 21-hydroxylase deficiency who had the haplotype HLA-B14,DR1 contained a hybridizing fragment 3700 base pairs long, indicating the presence of the valine-281 mutation in the CYP21B gene. In contrast, unaffected subjects and one patient with nonclassic deficiency who did not have HLA-B14,DR1 had no evidence of this mutation. We conclude that the mutation in codon 281 is a consistent molecular genetic marker for nonclassic 21-hydroxylase deficiency associated with HLA-B14,DR1.

Aldosterone Synthesis in Salt-Wasting Congenital Adrenal Hyperplasia with Complete Absence of Adrenal 21-Hydroxylase

BACKGROUNDnCongenital adrenal hyperplasia due to 21-hydroxylase deficiency is a disorder of cortisol and aldosterone biosynthesis that results from mutations in the CYP21 gene encoding the adrenal 21-hydroxylase P-450c21. It can cause severe salt wasting in newborns that requires long-term treatment with glucocorticoids and mineralocorticoids. We describe a spontaneous partial recovery from this disorder in a 19-year-old woman who had discontinued treatment.nnnMETHODSnWe measured plasma and urinary levels of adrenal hormones, plasma renin activity, and sodium balance longitudinally in the patient and four other patients in whom adrenal hyperplasia had been diagnosed in infancy and in whom DNA analysis had predicted a complete absence of functional P-450c21. The ratio of plasma renin activity to urinary aldosterone was used as a measure of the response of the adrenal zona glomerulosa. Two patients underwent intravenous infusion of [3H]progesterone for the measurement of extraadrenal production of 21-hydroxylated precursors of aldosterone.nnnRESULTSnThe patient who had discontinued her medication excreted a normal amount of aldosterone (20.0 nmol per square meter of body-surface area per day) while following a diet low in sodium. Her ratio of plasma renin activity to urinary aldosterone-18-glucuronide excretion was 1.7 after three days of sodium restriction, as compared with a ratio of 4.7 at the age of nine years (normal range, 0.03 to 0.1). The percentage of extraadrenal conversion of progesterone to deoxycorticosterone was low. The four other patients had variable responses to sodium restriction after the neonatal period (range for plasma renin activity:urinary aldosterone-18-glucuronide, 1.9 to 19.4).nnnCONCLUSIONSnAlthough patients with salt-wasting 21-hydroxylase deficiency have functionally equivalent mutations in their CYP21 genes, they may vary from one another and over time in their ability to produce mineralocorticoids. This variation may be attributable to another adrenal enzyme with 21-hydroxylase activity.

Investigation of the mechanism of hypertension in apparent mineralocorticoid excess

Apparent mineralocorticoid excess (AME) is a rare form of low renin hypertension caused by deficiency of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), the enzyme responsible for conversion of cortisol to the bio-inactive metabolite, cortisone. This results in prolonged cortisol half-life, activation of type I (mineralocorticoid) receptors by cortisol, sodium and fluid retention, and consequent childhood-onset hypertension. The cortisol secretion rate is low, perhaps due to cortisols binding to type II (glucocorticoid) receptors and suppressing corticotropin secretion. Patients with AME thus lack stigmata of Cushings syndrome. To evaluate any potential contribution of the type II (glucocorticoid) receptor to the development of hypertension in AME patients, we administered RU486, a steroid analogue that acts as a pure type II receptor blocker. Selective glucocorticoid receptor blockade did not decrease blood pressure in our patient; instead, a significant increase in average blood pressure was observed (125.1 +/- 1.7 pre-RU486 v 144.7 +/- 1.2 during RU486 treatment, P = .0001). We conclude that the type II receptor does not contribute to the development of hypertension in patients with AME.

FAILURE OF STEROID REPLACEMENT TO CONSISTENTLY NORMALIZE PITUITARY FUNCTION IN CONGENITAL ADRENAL HYPERPLASIA : HORMONAL AND MRI DATA

Exogenous glucocorticoid replacement in patients with congenital adrenal hyperplasia (CAH), who due to an adrenal 21-hydroxylase enzyme deficiency are unable to produce endogenous glucocorticoids, is aimed at normalizing hypothalamic-pituitary-adrenal function. Excess androgen production by the adrenals is thus decreased. Despite standard glucocorticoid replacement doses (12.5-40 mg, 10.5-27 mg/m2/day hydrocortisone equivalents) 4 of 7 patients ranging in age from 14 to 33 years had abnormalities of the pituitary on MRI. Three appeared to have microadenomas and 1 had an empty sella. Five (3 salt wasters, 2 simple virilizers) of these 7 patients had 60-min p.m. ovine corticotropin-releasing hormone (oCRH) stimulation studies. The mean (logarithm) area under the ACTH curve for 0-60 min after oCRH stimulation was significantly greater in patients than controls (p < 0.0001). Mean ACTH at each time point before and after oCRH stimulation was similarly greater in patients than controls (p < 0.05). Two of these patients had pituitary microadenomas, 1 had an empty sella; all 3 were salt wasters. Despite standard glucocorticoid replacement, adolescent and young adult patients with CAH tend to have high basal ACTH and ACTH hyperresponsiveness to oCRH, as well as structural abnormalities of the pituitary. The inevitable periods of under- and overexposure to glucocorticoids in CAH patients may over time cause abnormalities of the hypothalamic-pituitary-adrenal axis.

Polymorphism in the RD (D6S45) gene

SummaryThe RD (D6S45) gene in the class III region of the HLA major histocompatibility complex encodes a protein normally containing 24 consecutive basic-acidic dipeptide repeats. We determined the frequency of variations in the number of repeats by use of the polymerase chain reaction. Of 107 subjects 7 (3.3%) carried genes encoding 22 or 23 repeats. There was no difference in the frequency of such polymorphisms between normal individuals and those with systemic lupus erythematosus, a disease associated with other polymorphisms in the class III region of HLA. The frequency of polymorphisms in proteins with oligopeptide repeats may provide useful information concerning functional constraints on repeat number.