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The Departments of Obstetrics-Gynecology and Biochemistry (A.L., S.A.), University of Texas Southwestern Medical Center, Dallas, Texas 75390-9032; and Department of Pediatrics (I.A.H.), University of Cambridge School of Clinical Medicine, Cambridge, United Kingdom
Address all correspondence and requests for reprints to: Stefan Andersson, Ph.D., Department of Obstetrics-Gynecology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9032. E-mail: stefan.andersson{at}utsouthwestern.edu
Abstract
The 17ß-hydroxysteroid dehydrogenase (HSD) type 3 isozyme catalyzes the conversion of androstenedione to testosterone in the testis. Deleterious mutations in the HSD17B3 gene cause undermasculinization in genetic males attributable to impaired testosterone biosynthesis. Hence, a hallmark of this autosomal recessive disorder is a decreased plasma testosterone-to-androstenedione ratio. Here, a novel C268Y substitution mutation in exon 10 of the HSD17B3 gene, in a subject with 17ß-HSD 3 deficiency, is reported. Reconstitution experiments with recombinant protein reveal that substitution of tyrosine for cysteine at position 268 of 17ß-HSD type 3 abrogates the enzymatic activity. This finding brings to 20 the number of mutations in the HSD17B3 gene that cause male undermasculinization.
THE ROLE OF the 17ß-hydroxysteroid dehydrogenase (HSD) type 3 isozyme is to convert androstenedione to testosterone in the testes. Its gene, designated HSD17B3, contains 11 exons and is located on human chromosome 9q22 (1). 17ß-HSD 3 deficiency is an autosomal recessive disorder that manifests, in males, as undermasculinization characterized by hypoplastic-to-normal internal genitalia (epididymis, vas deferens, seminal vesicles, and ejaculatory ducts) but female external genitalia and the absence of a prostate. This phenotype is caused by inadequate testicular synthesis of testosterone, which, in turn, results in insufficient formation of dihydrotestosterone in the anlage of the external genitalia and prostate during fetal development. At the expected time of puberty, there is a marked increase in plasma LH and, consequently, in testicular secretion of androstenedione. Hence, a diagnostic hallmark of this disorder is a decreased plasma testosterone-to-androstenedione ratio. Significant amounts of the circulating androstenedione are, however, converted to testosterone, in peripheral tissues, by an unidentified member of the 17ß-HSD isozyme family, thereby causing virilization (2, 3). Women who are homozygous or compound-heterozygous for mutations that, in men, cause 17ß-HSD 3 deficiency are asymptomatic (4, 5).
To date, 19 mutations in the HSD17B3 gene that impair testosterone biosynthesis and cause male undermasculinization have been found. Fifteen of these molecular lesions are missense mutations, 3 are splice junction abnormalities, and 1 is a frame shift mutation (1, 2, 6, 7, 8). Here, we report 1 additional missense mutation in the HSD17B3 gene of a genetic male who was severely undermasculinized.
Subjects and Methods
Subjects
Patient designated 17HSD3-Cambridge 3. The patient was born with apparently normal female genitalia, after an uneventful pregnancy. The parents were of Pakistani origin and were first cousins. The infant presented, at age 3 weeks, with a right inguinal hernia. Physical examination revealed palpable gonads in both inguinal regions and a rather prominent clitoris. Biopsies were taken of both gonads, and the histology confirmed testicular tissue composed of Sertoli cells and spermatogonia in seminiferous tubules, with normal interstitial stroma. The karyotype was 46,XY, and an ultrasound examination of the pelvis showed no evidence of a uterus. There were four female siblings, each of whom had a 46,XX karyotype. Endocrine studies included a human (h)CG-stimulation test (1500 IU daily for 3 days) performed at 7 and 18 months of age. Basal LH and FSH at 18 months of age were 3.3 and 5.6 IU/L, respectively. Bilateral gonadectomy was performed at age 2 yr-3 months. A normal vas deferens was seen bilaterally, and an epididymis was identified on the right. The vagina was 2.5 cm in length; the slightly prominent clitoris was recessed. A genital skin biopsy was obtained to establish a fibroblast line. Histology of the testes showed Sertoli cells and spermatogonia in the seminiferous tubules, with normal interstitial stroma. Androgen receptor protein binding studies in genital skin fibroblasts were performed (9), and they revealed a Bmax (receptor concentration) of 0.4 fmol/µg DNA (normal, >0.3 fmol/µg DNA) and binding affinity of 0.25 nmol/L (normal, 0.08–0.17 nmol/L). All investigations were performed after approval from the Local Ethics Committee for the program of research on disorders of sex differentiation.
Mutation detection and expression analysis
Genomic DNA was extracted from white blood cells, and mutations in the HSD17B3 gene were analyzed by DNA sequencing of amplified exons using a thermostable DNA polymerase (2). Oligonucleotide-directed mutagenesis of the 17ß-HSD type 3 complementary DNA (cDNA) was performed using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. Complementary DNA expression in human embryonic 293 cells were performed using the Fugene 6 transfection reagent (Roche Diagnostics, Indianapolis, IN). Enzyme activity assays in intact cells were performed and analyzed in duplicates of 3 independent experiments, as described previously (6). Immunoblotting was performed using the monoclonal antibody MAb-C3–10 as described (6).
Results and Discussion
Initial endocrine analyses of a 46,XY prepubertal
undermasculinized male, designated 17HSD3-Cambridge 3, revealed a
plasma testosterone-to-androstenedione ratio of 0.25 and 0.29, after
hCG stimulation performed at 7 and 18 months of age, respectively.
These findings were indicative of the patient having 17ß-HSD 3
deficiency, given that the plasma testosterone-to-androstenedione ratio
normally is more than 0.8 after hCG stimulation, based on studies in 84
prepubertal undermasculinized patients without this genetic disorder
(10). The patient demonstrated a slight decrease in
androgen receptor binding affinity, as studied in genital skin
fibroblasts. We have previously reported similar subtle abnormalities
in androgen receptor binding characteristics in prepubertal subjects
with 17ß-HSD 3 deficiency (11), perhaps the result of
deficient androgen production in fetal and early postnatal life. The
changes, however, are not as conclusive as those observed in patients
with androgen receptor-mutant syndromes of androgen insensitivity
(9). DNA sequence analysis was performed on DNA fragments
amplified from genomic DNA of 17HSD3-Cambridge 3, who was found to be
homozygous for a novel C268Y substitution mutation attributable to a G
A transition in the second base of codon 268 in exon 10 of the
HSD17B3 gene (Fig. 1
).
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shows the results of a representative
time-course experiment in intact cells using 0.1 µmol/L
[3H]androstenedione as substrate. The mutated
protein was compared with the normal enzyme and vector transfected
cells (mock), and the data show that the C268Y mutation abolished the
17ß-HSD type 3 enzyme activity. To ensure equal expression of the
different recombinant proteins in the transfected cells, we performed
immunoblotting using a monoclonal antibody against the 17ß-HSD type 3
protein (Fig. 2B). To investigate whether the mutation altered the
enzyme’s affinity for the substrate, a time-course experiment
using 20 µmol/L [3H]androstenedione was
performed. However, no 17ß-HSD activity over background (mock
transfected cells) was observed with the Y268 protein (data not
shown).
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Acknowledgments
We thank Dr. Richard Stanhope and Mr. Philip Ransley for organizing patient samples for investigative studies.
Footnotes
1 This work was aided by Grant DK-52167 (to S.A. and A.L.) from the
NIH, and grants from the Tegger Foundation (to A.L.) and the Tore
Nilson Foundation for Medical Research (to A.L.), and the Wellcome
Trust (to I.A.H.) and Birth Defects Foundation (to I.A.H.). 
Received June 7, 2000.
Revised August 24, 2000.
Accepted October 13, 2000.
References
This article has been cited by other articles:
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  H. F. Escobar-Morreale, M. Luque-Ramirez, and J. L. San Millan The Molecular-Genetic Basis of Functional Hyperandrogenism and the Polycystic Ovary Syndrome Endocr. Rev., April 1, 2005; 26(2): 251 - 282. [Abstract] [Full Text] [PDF]
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