S Pang

Late onset 21-hydroxylase deficiency and HLA in the Ashkenazi population: A new Allele at the 21-hydroxylase locus

Abstract The congenital form of 21-hydroxylase deficiency (21-OH-def), which results in virilization at birth from intrauterine exposure to testosterone, results from the inheritance of an HLA-linked recessive disease allele from each parent a t the 21-OH locus (the 21-OH 0 allele). In this study we establish a relationship between intermediate and late onset 21-OH-def and HLA. In studies of five Ashkenazi families, we concluded that these syndromes can be caused by either of two combinations of genes at the 21-OH locus: They can occur in individuals who carry the 21-OH 0 allele on one baplotype and who in addition inherit a “susceptibility” 21-OH-def gene (21-OH 8 ) from their other parent (genotype = 21-OH 0 /21-OH 3 ; or they can occur in individuals who are homozygous for the “susceptibility” 21-OH-def allele (21-OH 8 /21-OH 3 ). The biochemical abnormality in late onset 21-OH-deficiency is characterized by elevated basline levels of plasma 17-hydroxyprogesterone (17-OH-P) and urinary pregnanetriol and/or relatively high 17-OH-P following ACTH stimulation. Although clinical symptoms are not always present in all family members with this biochemical abnormality, the abnormality appears to behave as a simple autosomal recessive trait that is linked to HLA. Among the five probands studied, eight of the eight haplotypes from five nonconsaguinous families assumed to carry the 21-OH 3 allele had the HLA antigens B14 and DRwl and also had the factor B (Bf) S (Slow) variant. The two remaining disease haplotypes were assumed from biochemical data to carry 21-OH 0 alleles. The results suggest that the biochemical abnormality in these syndromes is linked to HLA and that the 21-OH 3 allele has nonrandom association with the particular HLA haplotype B14, DRwl, BfS in the Ashkenazi population.

322 USEFULNESS OF PLASMA ANDROGEN CONCENTRATIONS FOR THE DIAGNOSIS OF VIRILIZING CONGENITAL ADRENAL HYPERPLASIA (VCAH) IN NEONATES AND YOUNG INFANTS

Plasma androgen concentrations, androstenedione, dehydroepiandrosterone (DHEA), testosterone (T) were measured in cord blood and from birth to 1 year-of-age in normal infants and in 4 male and 4 female infants ultimately proven to have VCAH. Cord blood androgen concentrations in two affected males did not differ from those of normal newborns (12 female, 16 males). Androstenedione in all affected infants was highly elevated while DHEA was elevated in only 3 infants. T was clearly elevated in all affected females. However, since the normal newborn male has high T concentrations (25-428), T concentrations in male infants with VCAH (60-295) were indistinguishable from normal. T in these infants decreased with glucocorticoid administration (blank-11) suggesting that the T was adrenal in origin and that testicular testosterone was suppressed in the untreated state. Conclusions: 1) cord blood androgen concentrations may not be diagnostic for VCAH; 2) plasma androstenedione is clearly a diagnostic hormone for VCAH in both sexes while T is useful only in female infants; and 3) the evidence of suppressed Leydig cell function in affected males suggests presence of negative feedback of the gonadstat in young male infants by excess adrenal steroids.

121 RECURRENCE OF CUSHING'S DISEASE AFTER PITUITARY DISEASE

We report a 10 year old female with Cushings disease with recurrent disease 5 yrs after successful pituitary irradiation. At presentation, bone age, skull films, head CT and visual fields were all normal. Basal serum F, urinary 17-OHCS, free cortisol (F), and 6β-hydrocortisol (6β-OHF) were elevated. Serum F lacked circadian variation and was partially suppressed by high dose dexamethasone (dex). She received pituitary irradiation with a cumulative dose of 4,000 rads over a 1 month period. Serum F, 17-OHCS, 6β-OHF, urinary free F returned to normal, growth velocity improved, and puberty ensued with menarche occurring 3 yrs after irradiation. However, lack of diurnal variation of F persisted. Despite radiotherapy, five years later she developed clinical and biochemical evidence of mild recurrent Cushings disease. Bone age, head CT, visual fields, and TRH, LHRH and L-DOPA/glucagon testing were all normal. Morning cortisol, 17-OHCS, and 6β-OHF were elevated and were partially suppressed only after high dose dex. The 8:00 PM ACTH level was elevated to 40 pcg/ml. After ovine CRF administration the maximum ACTH response was 46 pcg/ml; the ACTH concentration is increased and the absent ACTH response to CRF is abnormal. In patients with Cushings disease treated with radiotherapy, the ACTH response to CRF stimulation may not be reliably compared to that of normal controls.Conclusion: Therefore Cushings disease may recur despite successful pituitary irradiation. This suggests a hypothalamic CRF producing lesion as the primary lesion in Cushing s disease.

113 11|[beta]|-HYDROXYLASE DEFICIENCY-CONGENITAL DEFECT OR TUMOR

In 3 children with low renin hypertension and virilism, the hormonal profile distinguished the patient with an adrenal tumor producing DOC and androgens from the other 2 patients who had a genetic deficiency of steroid 11β-hydroxylase (11β-OHD). Clinical presentations were remarkably similar as were the baseline serum concentrations of deoxycorticosterone (DOC). The major difference in the baseline hormones consisted of very high DHEA levels in the tumor patient in contrast to very high androstenedione (Δ4) levels in the patients with the genetic defect of 11β-hydroxylation. The stimulation and suppression tests also distinguished the tumor patient. DOC did not stimulate with ACTH nor suppress with dexamethasone (dex). The PRA rose with dex suppression only in the patients with the genetic defect. Serum aldosterone was undetectable in all 3 patients in the baseline period.Conclusion: Though patients with adrenal tumors and those with a genetic deficiency of 11β-OHD have similar clinical presentations, similar PRA, aldosterone and DOC, the androgens and their steroid responses to ACTH and dex are clear distinguising features. (nd=not detectable).

480 ADRENAL CORTICAL FUNCTION IN CHILDREN WITH HYPOPITUITARISM SUGGESTS ACTH IS THE PITUITARY ADRENAL ANDROGEN STIMULATING TROPIC HORMONE

Baseline and ACTH stimulated (4OU IV infusion for 360 min) serum concentrations of deoxycorticosterone (DOC), cortisol (F), androstenedione (Δ4-A), dehy-droepiandrosterone (DHA), DHA-sulfate (DS), and aldosterone (aldo) in prepubertal children with isolated growth hormone deficiency (N=5) and in children with two or more pituitary hormone deficiencies (def) without ACTH def (N=5) were similar to those of normal prepubertal children whose age matched to bone age of hypopituitary children. However in children with two or more pituitary hormone def including ACTH def (N=5) there was a markedly low response of DOC, F,Δ4A, DHA and DS to ACTH stimulation but normal response of aldo. Hormonal response to ACTH stimulation in hGH treated hypopituitary children did not differ from the untreated hypopituitary (Hyp) children. Thus, adrenal androgen secretory function is normal in children with multiple or single pituitary hormone def providing ACTH secretion is normal, while in ACTH deficient hypopit children, adrenal androgen secretion is very low. These data suggest that ACTH is the tropic hormone involved in the maturation of adrenal androgen secretion.

CUSHING'S SYNDROME AND HIRSUTISM DUE TO A MALIGNANT OVARIAN TUMOR

Ovarian tissue is classically devoid of 11β-hydroxylase and 21-hydroxylase activity. We report a 19-year-old female who presented with Cushings syndrome, hirsutism and secondary amenorrhea and was found to have a metastatic ovarian tumor autonomously producing cortisol (F). ACTH levels were within the normal range. Peripheral levels of Δ4-androstenedione (Δ4-A) were markedly elevated. The tumor was only minimally responsive to ACTH and neither glucocorticoid nor androgen levels were suppressible with dexamethasone. This suggests that F production in the ovarian tissue was not under ACTH control. Upon removal of the tumor, androgen levels fell to within the normal range and serum F returned to a normal circadian pattern. Excess facial hair vanished and menses resumed within one month of tumor removal. Pathology was consistent with lipoid cell tumor of the ovary, but adrenocortical carcinoma arising in an ovarian-adrenal rest tumor could not be ruled out. The patient was therefore begun on monthly courses of chemotherapy with adriamycin, cis-platinum, and opDDD therapy. The hormonal results are tabulated below.We therefore report a metastatic ovarian tumor resulting in Cushings syndrome with autonomous cortisol secretion.

H-Y ANTIGEN DETECTED BY ENZYME LINKED IMMUNOSORBENT ASSAY |[lpar]|ELISA|[rpar]|

In a newborn (46,XX) with ambiguous genitalia and bilaterally descended gonads the presumptive diagnosis of true hermaphroditism (TH) was made using endocrinological methods and a newly developed ELISA system with monoclonal H-Y antibody. Evidence for cryptic testicular tissue was obtained by measuring male levels of testosterone in plasma (117 ng/dl). 170H progesterone levels were normal thus ruling out congenital adrenal hyperplaisa.ELISA for H-Y antigen involves the reaction of monoclonal H-Y lgG antibody (AB) and cell borne or soluble antigen (AG). After that a 2nd AB (conjugated to peroxidase enzyme) is added. The amount of color (optical density) measured in a densitometer is a function of the interaction of AB and AG and consequential reaction of perioxidase with substrate. The patient tested positive for H-Y antigen but levels were less than those found in normal males. H-Y positive phenotypes in XX TH and XX male sex reversal obtained with this technique are consistent with the notion that the two conditions are variants of the same X-linked disorder and that the morphologic difference between the two may be related to the degree of inactivation of the X chromosome bearing the mutant gene. TH was confirmed at surgery when ovotestes were found. Conclusion: H-Y testing using ELISA technology can be performed rapidly with small amounts of blood (5 ml). Thus this assay is particularly useful in the evaluation of newborn infants with ambiguous genitalia where quick diagnosis is essential.

VIRILIZATION IN A 45, X/46, Xr(X)/46, Xde1(X)(p21: ;q11:) FEMALE

A 14 1/2 year old female with short stature, bone age 12, Tanner I breasts, pubic hair and clitoromegaly (3.5 x 1.5 cm) was studied. Lymphocyte karyotypes were 45,X/46,Xr(X)/46,Xdel(X) (p21:;q11). Abdominal and pelvic sonogram and CTT were normal. Elevated and fixed levels of serum testosterone were noted.There was indication of expression of H-Y antigen in blood lymphocytes evaluated in the ELISA. Bilateral streak gonads were surgically removed and contained multiple clusters of Leydig cells. Postoperatively the T level decreased to 13 ng/dl.Thus, in the patients gonads there was evidence of functional Leydig cells in the absence of a Y line. Presence of H-Y antigen may define a group of patients with gonadal dysgenesis who are at risk for development of gonadoblastoma.

Genotyping for 21-hydroxylase deficiency: one or two genes?

We have devised nomograms relating the baseline and ACTH stimulable levels of 17-OHP, Δ 4-androstenedione and testosterone for genotyping 21-hydroxylase deficiency. The nomograms provide a method for classifying the patient with congenital, late onset or cryptic 21-hydroxylase deficiency as well as classifying the heterozygotes for each of these disorders. In addition, the subject predicted by HLA genotyping to be genetically unaffected can also be classified by these nomograms. Further the nomograms permit us to obtain evidence for genetic recombination between HLA and the 21-hydroxylase locus. For example a patient predicted by initial HLA genotyping to be unaffected was classified by the nomogram to be a heterozygote. When HLA-DR typing was performed an informative maternal HLA A:DR recombination was discovered. This recombination explained the heterozygote response of this subject. In another family a maternal DR:GLO recombination was found in an asymptomatic sister who was HLA identical to the patient with late onset 21-hydroxylase deficiency. Although most recombinants have mapped the gene for 21-hydroxylase between B and DR, this DR:GLO recombination presents evidence that there may also be a 21-hydroxylase locus between the DR-GLO loci. The nomograms thus provide a powerful tool to determine the 21-hydroxylase genotype by hormonal testing and assist in mapping the gene for 21-hydroxylase deficiency.

Late-Onset, Cryptic and Classical 21-OH Deficiency: Allelic Variants

HLA genotyping and hormonal studies in 9 females with non-classical steroid 21-hydroxylase deficiency (AAH) indicate that this disorder is due to an autosomal recessive gene linked to HLA, similar to classical and cryptic 21-hydroxylase deficiency (21-OH def). They had normal genitalia at birth and presented between 9 mos to 16 yrs with varying degrees of virilization. Hormonal studies of the families revealed 2 fathers and their HLA identical sisters with 21-OH def. The remaining parents and the sibs sharing one HLA haplotype with the AAH patient responded to ACTH stimulation as heterozygotes for classical or cryptic 21-OH def. Five sibs who were HLA identical to their affected sib also had findings diagnostic of 21-OH def. The hormonal response to ACTH of the patients with AAH and their HLA identical sibs was similar to that of patients with cryptic 21-OH def. Thus, individuals with these non-classical forms of 21-OH def and similar hormonal findings present with a clinical spectrum ranging from an asymptomatic deficiency to precocious pubic hair, acne, tall stature and advanced bone age, hirsutism, clitoromegaly and menstrual irregularities. The results of these studies support the concept that AAH, similar to classical and cryptic 21-OH def is due to an HLA linked autosomal recessive gene and that these disorders are due to allelic variants at the locus of steroid 21-hydroxylase.