NOTE
21-Hydroxylase Deficiency Presenting as Massive Bilateral Adrenal Masses in the Seventh Decade of Life
KATSUMI ABO, KIMIAKI SUMINO, HISAHIDE NISHIO, TOSHIKI HOZUMI*, YOSHIHIKO ISHIDA*,
KENJI FUJIEDA ** , TOSHIHIRO TAJIMA ** AND TSUTOMU KAZUMI ***
Department of Public Health, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan
* Department of Medicine, Hyogo Medical Center for Adults, 13-70 Kitaozi-cho, Akashi 673-0021, Japan
** Department of Pediatrics, Hokkaido University School of Medicine, N15 W7, Kita-ku, Sapporo 060-0815, Japan
*** Department of Medicine, Hyogo Rehabilitation Center Hospital, 1070 Akebono, Nishi-ku, Kobe 651-2134, Japan
Abstract. A 72-year-old woman was found to have massive bilateral adrenal masses on computed tomography and was diagnosed with 21-hydroxylase deficiency (21-OHD) based on endocrinological findings. Physical examination revealed no abnormalities except markedly short stature. She was diagnosed with 21-OHD because she had an elevated serum 17a-hydroxyprogesterone (17-OHP) level which significantly decreased in response to dexamethasone. Percutaneous CT-guided biopsy and later autopsy confirmed that the adrenal masses were due to adrenocortical hyperplasia. Analysis of the CYP21 gene revealed that the patient was a compound heterozygote for the Ile- 172→Asn mutation in exon 4 and the 8-bp deletion in exon 3. Simple virilizing 21-OHD (SV) would be predicted from this genotype. She had few symptoms associated with 21-OHD except for markedly short stature, but the serum 17-OHP level was higher than that of typical nonclassical form of 21-OHD and near to that of typical SV. This finding was confirmed by analysis of the CYP21 gene. From these results, we report that when adrenal masses are incidentally detected, 21-OHD should be ruled out to avoid excessive examination and surgery on the suspicion of adrenal carcinoma.
Key words: Bilateral adrenal masses, 21-Hydroxylase deficiency, CYP21 gene
(Endocrine Journal 46: 817-823, 1999)
21-HYDROXYLASE deficiency (21-OHD) is among the most common inherited disorders and accounts for 90-95% of congenital adrenal hyperplasia (CAH) cases [1, 2]. The two major classic disease pheno- types of 21-OHD are the salt wasting (SW) and simple virilizing (SV) forms. Nonclassical 21-OHD (NC), a milder form of the disease, is distinguished from the classical 21-OHD. Most untreated CAH patients including 21-OHD patients have adrenal masses [3, 4]. Recently, several cases of adrenal masses have been discovered incidentally on com- puted tomography (CT) and further work-up showed
they had 21-OHD [5, 6]. The 21-hydroxylase en- zyme is encoded by the functional CYP21 gene, which is nearly identical with CYP21P pseudogene. Recent studies suggest that the various phenotypes of 21-OHD correspond with particular mutations in the CYP21 gene [7-9], whereas it has been reported that the genotype does not always predict the phenotype [9, 10]. Here we report a case of 21-OHD diagnosed when bilateral adrenal masses were discovered on CT at age 72. We also discuss the diagnosis of adrenal mass and the relationship between the 21-OHD phenotype and CYP21 genotype.
Case report
A 72-year-old woman was referred to the Depart- ment of Medicine, Hyogo Medical Center for Adults
in June 1994 because of fever of unknown origin. Upper abdominal CT revealed bilateral massive adrenal masses, and she was admitted to the hospital for extensive examination. She denied any history of repeated vomiting or hospitalizations as an infant or child. She was 125 cm tall at age 12, when she stopped growing. She developed breasts at age 14 and pubic hair at age 15. She began to menstruate at age 17 and her menses were irregular. She was married at age 22 but was never pregnant.
Physical examination revealed that she was 128 cm tall and weighed 37 kg. Her blood pressure was 128/70 mm Hg, pulse rate was 60/min and regular, and body temperature was 37.8℃. She had sparse head hair but normal pubic hair. Slight hyperpig- mentation was found in the pubic region. Exami- nation of the genitalia revealed no clitoromegaly, scrotalization of the labial folds, or any other ab- normalities.
Baseline laboratory evaluation showed the follow- ing values: leukocyte, 8800/pl; erythrocyte, 381 × 104/pl; Hb, 10.4 g/dl; Hct, 31.4%; Na, 138 mEq/1; K, 4.2 mEq/1; CRP, 7.3 mg/dl. Karyotype was 46XX. HLA haplotype was A24 (9), A31 (19), B60
(40), B51 (5), Cw3, DR2, DR6. The baseline cortisol and aldosterone levels were normal. The baseline ACTH level was slightly elevated (Table 1-a). Compared with the normal circadian fluctuation of ACTH and cortisol levels, the cortisol and ACTH levels at 11 p.m. in this patient were both slightly elevated (Table 1-b). Dexamethasone completely suppressed serum cortisol and ACTH. The 17a- hydroxyprogesterone (17-OHP) level was extremely high at baseline, and increased in response to ACTH stimulation test (Table 2-a). On ACTH stimulation test, serum cortisol increased within a twofold higher level from the basal level. These findings suggested slight adrenocortical insufficiency and therefore the levels of various adrenocortical steroids were meas- ured (Table 1-a). The patient had high levels of urinary 17-ketosteroids (17-KS), pregnanetriol, and 11-deoxy-ketogenicsteroids/11-oxy-ketogenicsteroids ratio (11-deoxy-KGS/11-oxy-KGS ratio), and all were significantly suppressed in response to dex- amethasone suppression test (Table 2-b).
Upper abdominal CT and magnetic resonance im- aging (MRI) showed a left adrenal gland of 53 × 18 mm in size and a right adrenal gland of 32× 23
| (a) Hormone | (normal range) | |||
|---|---|---|---|---|
| Serum | ACTH | (pg/ml) | 70 | (6.1-55) |
| Progesterone | (ng/ml) | 9.7 | (0-5.3) | |
| Deoxycorticosterone | (ng/ml) | 0.25 | (0.03-0.33) | |
| Aldosterone | (pg/ml) | 120 | (30-160) | |
| 17-OHP | (ng/ml) | 65 | (0.1-3.3) | |
| 11-deoxycortisol | (ng/ml) | 0.61 | (0.11-0.60) | |
| Cortisol | (µg/dl) | 9.7 | (4.4-17.4) | |
| DHEA-s | (ng/ml) | 1630 | (400-3500) | |
| 44-androstendione | (ng/ml) | 14 | (0.6-5.0) | |
| testosterone | (ng/ml) | 2.3 | (0.1-0.9) | |
| estradiol | (pg/ml) | 50 | (1.1-61.7) | |
| Urine | 17-OHCS | (mg/day) | 6.6 | (2.4-5.8) |
| 11-deoxy-KGS | (mg/day) | 67.3 | (1.2-2.9) | |
| 11-oxy-KGS | (mg/day) | 69.9 | (7.0-16.8) | |
| pregnanetriol | (mg/day) | 34.4 | (0.02-0.83) | |
| 17-KS | (mg/day) | 47.0 | (3.3-13.8) | |
| (b) Circadian fluctuation of serum ACTH and cortisol | ||||
| 8 a.m. | 4 p.m. | 11 p.m. | ||
| ACTH | (pg/ml) | 70 | 31 | 47 |
| Cortisol | (µg/dl) | 10 | 6.3 | 11 |
| (a) ACTH stimulation test* | ||||||
| (min) | 0 | 30 | 60 | |||
| Corsitol | (µg/dl) | 9.7 | 14 | 14 | ||
| 17-OHP | (ng/ml) | 65 | - | >200 | ||
| (b) Dexamethasone suppression test* | ||||||
| (day) | 0 | 1 | 2 | 3 | 4 | |
| Serum | ||||||
| ACTH | (pg/ml) | 47 | <5.0 | <5.0 | <5.0 | <5.0 |
| Cortisol | (µg/dl) | 8.3 | <1.0 | <1.0 | <1.0 | <1.0 |
| 17-OHP | (ng/ml) | 110 | - | 2.7 | - | - |
| Urine | ||||||
| Pregnanetriol | (mg/day) | 27.2 | - | - | - | <1.0 |
| 17-KS | (mg/day) | 46.1 | 25.6 | 9.8 | 4.6 | 4.5 |
| 11-deoxy-KGS/11-oxy-KGS | 1.15 | 0.88 | 0.86 | 0.61 | - | |
*A single intravenous injection of Tetracosactide 0.25 mg
*Oral administration of Dexamethasone 2 mg/day for 2 days, followed by oral administration of 8 mg/day for another 2 days
a)
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b)
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mm in size (Fig. 1). Percutaneous CT-guided biopsy of the left adrenal gland revealed no malignancy. Adrenal 131I-Adosterol scintigraphy showed increased uptake by the bilateral adrenal glands. Angio- graphy showed relatively regular tumor staining of multiple nodular states. Since the urinary 17-KS level was considerably high, blood samples were obtained from the left and right adrenal veins and two points on the inferior vena cava, that is, the central side of the turning point of the right adrenal vein and the peripheral side of the turning point of the left renal vein. The dehydroepiandrosterone- sulphate (DHEA-S), androstendione and testoster- one levels in the blood samples obtained from these four points were nearly equal.
After obtaining informed consent from the patient, a genomic DNA sample was extracted from her blood leukocytes and the CYP21 gene was ana- lyzed. Large deletions were not detected in the CYP21 gene by Southern blot analysis method described previously [11].
To identify point mutations, polymerase chain
reaction (PCR)-restriction enzyme analysis was per- formed, as described previously [11]. Among the known deleterious mutations of the CYP21 gene causing 21-OHD, we analyzed the intron 2 mutation (C or A nucleotide→G nucleotide), exon 4 mutation (Ile-172→Asn, E4IN), exon 7 mutation (Val-281 →Leu) and exon 8 mutation (Gln-318→Stop). The primer pairs which we used to amplify specific regions of the CYP21 gene are shown in Table 3. Each of the PCR products was digested with the appropriate restriction enzyme, according to the manufacturer’s recommended protocols (Boehringer Mannheim, Mannheim, Germany). After digestion, the reaction mixture was electrophoresed on a 2.5% agarose gel for 45 min at 100 mV. Only the hetero- zygous E4IN mutation was detected by PCR-res- triction enzyme analysis (Fig. 2).
To further analyze the CYP21 gene of the patient, DNA sequence analysis was performed according to the chain-termination method described previously [11]. For PCR followed by direct sequence analysis, the following primer pairs were used: G-H, I-J
| Primer | Sequence | Location |
|---|---|---|
| A | 5'-TGGGGCATCCCCAATCCAGGTCC-3' | 524-546 |
| B | 5'-ACCAGCTTGTCTGCAGGAGGA T-3' | 677-656 |
| C | 5'-TTCTCTCTCCTCACCTGCAGCATC G-3' | 974-998 |
| D | 5'-CTGCATCTCCACGATGTGATCCCTC-3' | 1393-1369 |
| E | 5'-GATCACATCGTGGAGATGCAGCTG-3' | 1373-1396 |
| F | 5'-TGGGCCGTGTGGTGCGGTGGGGCAA-3' | 2153-2129 |
| G | 5'-TCGGTGGGAGGGTACCTGAA-3' | -122 -- 103 |
| H | 5'-AGCAGGGAGTAGTCTCCCAAGGA-3' | 722-700 |
| I | 5'-AAGGTCAGGCCCTCAGCTGCCTTCA-3' | 606-630 |
| J | 5'-ATGTGCACGTGCCCTTCCAGGAG-3' | 1690-1668 |
| K | 5'-AGAAGAGGGATCACATCGTGGAGAT-3' | 1365-1389 |
| L | 5'-GTGGGCGCCTTGGAGGTTCGGAAT-3' | 2301-2278 |
| M | 5'-TCCTTGGGAGACTACTCCCTGCT-3' | 700-722 |
Primers A, B, C, D, E and F were used for PCR-restriction enzyme analysis. Primer pairs A-B and C-D were used for PCR-restriction enzyme analysis of the intron 2 mutation (I 2 g) and the exon 4 mutation (E4IN), respectively. In primer B and C, the T and G nucleotides at the 3’ end were mismatched to the respective nucleotides in the CYP21 gene sequence to introduce the Sau3AI and TaqI res- triction sites by PCR into the mutant sequence, respectively. Primer pair E-F was used for PCR-restriction enzyme analysis of the exon 7 mutation (E7VL) and the exon 8 mutation (E8 non). Primers G, H, I, J, K and L were used for PCR-direct sequence analysis. The primer pair M-J was used to analyze whether or not the E4IN mutation and the exon 8 deletion (E3gdl) are present in the same allele. “Location” indicates the nucleotide number as described previously [12].
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and K-L (Table 3). A heterozygous 8-nucleotide (nucleotide number 707-714 [12]) deletion in exon 3 (E3gdl) was detected by DNA sequence analysis (Fig. 3).
To detect which allele carries the E4IN mutation and the E3gdl deletion, another PCR-restriction en- zyme analysis was performed using primer pair M-J. Primer pair M-J was set up not to amplify the allele which carries the E3gdl deletion. The E4IN mutation was detected by this analysis, indicating that the E4IN mutation and the E3gdl deletion are present on different alleles of the CYP21 gene.
After admission, all inflammatory findings disap- peared without any treatment. Since the patient was diagnosed with 21-OHD, treatment with 0.5 mg/day
oral dexamethasone was started, and the ACTH level was suppressed to less than 5.0 pg/ml. Later, the dosage of dexamethasone was decreased to 0.5 mg every two days and she was discharged. In May 1995, a metastatic liver tumor was detected on CT. Later on her tumor was confirmed as pancreatic cancer. She died in February 1996. The size of the bilateral adrenal glands on CT did not change until her death. The autopsy revealed a left adrenal gland of 29 g measuring 80×60×18 mm, and a right adrenal gland of 40 g measuring 90x 70× 20 mm. The histologic diagnosis of the bilateral masses was adrenocortical hyperplasia.
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Discussion
Our patient was diagnosed with 21-OHD because she had elevated levels of serum 17-OHP, urinary pregnanetriol, and urinary 11-deoxy-KGS/11-oxy- KGS ratio, all of which decreased in response to dexamethasone. She denied any history of salt- wasting episodes and had few symptoms except for markedly short stature which is considered to be associated with 21-OHD. The clinical diagnosis is commonly confirmed by analysis of the CYP21 gene [7-9]. Our patient was a compound heterozygote for the E4IN mutation and the E3gdl deletion. The E3gdl deletion renders the gene nonfunctional by
generating premature termination [8], and this dele- tion is often detected in the CYP21 gene of SW patients [13]. The E4IN mutation reduces 21- hydroxylase enzyme activity to less than 10% of normal activity [8], and this mutation is often found in the CYP21 gene of SV patients [9, 14]. Therefore, a compound heterozygote for these two nucleotide alterations should result in the SV phenotype, con- sidering that 21-OHD is an autosomal recessive dis- ease. Speiser et al. investigated the relationship be- tween 17-OHP level and phenotype of 21-OHD, and reported that the basal level of serum 17-OHP in SV patients is usually > 300 nmol/1 (99 ng/ml), while NC patients have a lower basal level of 17-OHP [9]. In our case, the serum 17-OHP level was higher than that of typical NC and near to that of typical SV.
As most untreated CAH patients including 21- OHD patients have adrenal masses [3, 4], our patient was diagnosed as having 21-OHD when bilateral adrenal masses were incidentally discovered on CT. Jaresch et al. examined 22 patients with homozygous CAH (of which 20 patients had 21-OHD) who were under replacement therapy with hydrocortisone, and reported that adrenal tumors were detected in 82% of the patients on CT [4]. Twenty siblings of the 22 homozygous CAH patients were diagnosed with heterozygous CAH (of which 19 siblings had 21- OHD), and adrenal tumors were detected in 45% of the siblings with heterozygous CAH. Therefore, in order to detect NC or heterozygous CAH, patients with adrenal masses should have an ACTH stimu- lation test unless the cause of the mass is otherwise evident [5]. Small incidentalomas are markedly less likely to be carcinomas than large incidentalomas. Therefore, Ravichandran et al. mentioned that CAH patients with smaller incidentalomas should be fol- lowed both clinically and with periodic CT scans, and that biopsy or surgery need not be routine [5]. In our patient, as both adrenal masses exceeded 3 cm in diameter and the inner part of the masses showed heterogeneity, adrenocortical carcinoma could not be completely ruled out. Therefore, percutaneous CT- guided biopsy was performed. Efforts should be made to check for 21-OHD in patients with adrenal masses discovered incidentally, to avoid excessive examination and surgery.
References
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