Proc. Natl. Acad. Sci. USA Vol. 85, pp. 8151-8155, November 1988 Genetics Deletion of the steroid-binding domain of the human androgen receptor gene in one family with complete androgen insensitivity syndrome: Evidence for further genetic heterogeneity in this syndrome (/46,XY karyotype/male pseudohermaphroditism/testicular feminization) TERRY R. BROWN*t, DENNIS B. LUBAHN*, ELIZABETH M. WILSONO§, DAVID R. JOSEPH*¶, FRANK S. FRENCHt, AND CLAUDE J. MIGEON* *Department of Pediatrics, Pediatric Endocrine Laboratories, Johns Hopkins University School of Medicine, Baltimore, MD 21205; and Departments of SPediatrics, §Biochemistry, and Physiology, Laboratories for Reproductive Biology, University of North Carolina, Chapel Hill, NC 27599 Communicated by John W. Littlefield, August 1, 1988

ABSTRACT The cloning of a cDNA for the human andro- In the complete form ofAIS, affected subjects present with gen receptor gene has resulted in the availability of cDNA completely external genitalia, but with a short probes that span various parts of the gene, including the entire and absent Mullerian ducts; with normal-size testes located steroid-binding domain and part of the DNA-binding domain, in the abdominal or inguinal area, but with hypoplastic as well as part of the 5' region of the gene. The radiolabeled Wolffian ducts; with normal female secondary-sex charac- probes were used to screen for androgen receptor mutations on teristics at , but usually sparse or absent pubic and Southern blots prepared by restriction endonuclease digestion body hair (2, 3, 9). In adults, serum testosterone and lutropin of genomic DNA from human subjects with complete androgen concentrations are at or above the normal adult male range insensitivity syndrome (AIS). In this investigation, we consid- (16). Determination of androgen receptor binding in cultured ered only patients presenting complete AIS and with the genital skin fibroblasts has demonstrated the genetic heter- androgen receptor (-) form as the most probable subjects to ogeneity ofcomplete AIS, some patients having undetectable show a gene deletion. One subject from each of six unrelated binding, or the so-called receptor (-) form, whereas others families with the receptor (-) form of-complete AIS and 10 have quantitatively normal binding, termed the receptor (+) normal subjects (6 and 4 males) were studied. In the 10 form (15, 17, 18). normal subjects and in 5 of the 6 patients, identical DNA Cloning and partial analysis of human cDNA encoding the restriction fragment patterns were observed with EcoRI and androgen receptor gene has been reported (19, 20). Various BamHI. In one affected individual, a partial deletion of the fragments of the cDNA have been used in the present study to probe the genome of six unrelated patients with the androgen receptor gene involving the steroid-binding domain receptor ( -) form ofcomplete AIS. Five patients had normal was detected. Analysis of other members of this family con- restriction fragment patterns with EcoRI and BamHI endo- firmed the apparent gene deletion. Our data provide- direct nucleases, suggesting a point mutation (or small, undetected proof that complete AIS in some families can result from a deletion) of their androgen receptor gene. In one subject and deletion of the androgen receptor structural gene. However, her affected sibling, a partial deletion of the gene was other families do not demonstrate such a deletion, suggesting observed, demonstrating further genetic heterogeneity in that point mutations (or small, undetectable deletions) may also receptor (-), complete AIS. A preliminary report of this result in the receptor (-) form ofcomplete AIS, adding further work has been presented. 11 to the genetic heterogeneity of this syndrome. MATERIALS AND METHODS Male pseudohermaphroditism in humans is defined as a condition of incomplete masculinization of the fetal external Experimental Subjects. Informed written consent was ob- genitalia in a karyotypically normal 46,XY individual (1). tained from each subject according to Johns Hopkins Insti- Among the multiple genetic disorders that result in male tutional Guidelines for Clinical Investigation. We have stud- pseudohermaphroditism are a group of abnormalities of the ied 10 normal subjects (6 females and 4 males) and 1 affected androgen target cells, including Sa-reductase deficiency and individual from each of six unrelated families with the androgen insensitivity (2, 3). The androgen insensitivity receptor (-) form of complete AIS. Several members of one syndrome (AIS) is an X chromosome-linked disorder (4, 5) of the families were also studied. The pedigree of this family resulting from impairment of the biological actions of andro- is shown in Fig. 1. The father is deceased and the mother (1-2) genic hormones related to defects in the intracellular andro- was shown to be heterozygous for the androgen receptor gen receptor (6-8). Phenotypic expression is quite variable gene mutation (4). Dizygotic twins, II-3 and II-4, as well as ranging from complete female phenotype to male genitalia a sibling, 11-7, are phenotypic females including female with mild (9, 10). Recent evidence suggests that external genitalia and breast development, without clitoral the syndrome includes some rare cases of phenotypically enlargement or posterior labial fusion. They have only sparse normal males with infertility (11, 12). The heterogeneity in amounts of pubic and axillary hair. In subjects II-3 and 11-4, is due the plasma testosterone concentrations were 661 and 637 ng/dl phenotypic expression to variety of androgen = + levels receptor defects, some of which are detectable by biochem- (normal male 550 150 ng/dl) and serum lutropin ical methods (13-15). Abbreviation: AIS, androgen insensitivity syndrome. tTo whom reprint requests should be addressed. The publication costs of this article were defrayed in part by page charge IBrown, T. R., Lubahn, D. B., Wilson, E. M., Joseph, D. R., payment. This article must therefore be hereby marked "advertisement" French, F. S. & Migeon, C. J., 78th Annual Meeting of the in accordance with 18 U.S.C. §1734 solely to indicate this fact. Endocrine Society, June 8-11, 1988, New Orleans, abstr. 28.

8151 Downloaded by guest on September 28, 2021 8152 Genetics: Brown et al. Proc. Natl. Acad. Sci. USA 85 (1988) I fibroblasts, cells from 10 to 16 100-mm culture plates were I 2 trypsinized and removed by repeated pipetting with Tris- buffered saline (10 mM Tris HCI, pH 7.4/0.9% NaCl). Cells were collected by centrifugation at 800 x g for 5 min at 4°C. The cells were resuspended in 10 ml of Tris-buffered saline and resedimented. The cells were resuspended in 10 ml of a R solution of 50 mM Tris HCl (pH 7.8), 0.15 M NaCl, and 5 mM 4 5 7 59 EDTA, incubated in the presence of proteinase K (1-2 mg) FIG. 1. Pedigree of a family with receptor (-) form of complete for 15 min at 650C, and then incubated for an additional 12- AIS. The father (I-1) is deceased; the mother (1-2) is an obligate 16 hr at 370C with shaking. This solution was extracted three heterozygote of this X-linked trait (solid circle inside open circle); times with an equal volume (10 ml) of phenol/chloroform, 1: and the three affected 46,XY subjects (solid symbols) are dizygotic 1 (vol/vol), and twice with chloroform/isoamyl alcohol, 24: twins, 11-3 and 11-4, and a sibling, 11-7. 1 (vol/vol). DNA was precipitated from the aqueous phase by the addition of 5 ml of 7.5 M ammonium acetate and 37.5 ml were 36.4 and 57.0 milliinternational units/ml (normal male of ice-cold ethanol followed by centrifugation at 10,000 x g = 8.9 + 3.5 milliinternational units/ml), respectively (pa- for 15 min at 4°C. The supernatant was decanted and the tients 3 and 4 of ref. 21). receptor DNA pellet was washed with 5 ml of 80% (vol/vol) ethanol. binding was absent in skin fibroblasts cultured from subjects DNA was dissolved in 5 ml of 10 mM Tris HCl (pH 7.4) plus 11-3 and II-4 but present at normal levels in a sexually normal 1 mM EDTA (TE) by shaking at 37°C for 12-16 hr. The brother (11-5), in two younger sisters (II-8 and II-9), and in solution was treated with RNase A (100 ,ug/ml) for 30 min at their mother (I-2), an obligate heterozygote (4). Five subjects 37°C and reextracted twice with 5 ml of phenol/chloroform, from this family, 1-2, II-2, 11-3, II-4, and II-9, are included in 1:1 (vol/vol), once with chloroform, and once with chloro- this study. form/isoamyl alcohol, 24:1 (vol/vol). DNA was precipitated Hybridization Probes. Two of the hybridization probes for from the aqueous phase by the addition of 0.5 ml of 3 M the androgen receptor, hAR-1 and hAR-2, were derived from sodium acetate and 12.5 ml of ice-cold ethanol followed by clone ARHFLlH-X obtained from a human foreskin fibro- centrifugation at 10,000 x g for 15 min at 4°C. The pellet was blast cDNA library as described by Lubahn et al. (20). washed with 5 ml of ice-cold 80% (vol/vol) ethanol and ARHFLlH-X contains a portion ofthe DNA-binding domain recentrifuged. DNA from leukocytes and skin fibroblasts was as determined from nucleotide sequence data. When cloned incubated in TE buffer at 37°C with shaking until dissolved into the expression vector pCMV, ARHFLlH-X also syn- and the concentration of each sample was determined spec- thesizes a protein that binds dihydrotestosterone specifically trophotometrically. and with high affinity. As shown in Fig. 2, hAR-1 is a 718-base DNA (8-10 jig) was digested for 16-20 hr with various pair (bp) cDNA fragment containing a portion of the DNA- restriction endonucleases according to the manufacturer's binding domain at its 5' terminus and extending toward the 3' recommendations for buffer and temperature. DNA frag- steroid-binding domain. hAR-2 is a 490-bp cDNA contiguous ments were separated by agarose gel electrophoresis [1% with hAR-1 and contains the extreme 3' coding sequence and (wt/vol)], transferred in alkaline buffer to GeneScreenPlus some of the 3' noncoding region. hAR-3 is a 575-bp cDNA (DuPont/NEN), and neutralized on the membrane. The mem- fragment derived from clone gtll ARHEL1 that was isolated brane was prehybridized overnight at 42°C in a solution con- from a human epididymal cDNA library and localized on the taining 50% (vol/vol) deionized formamide, 5 x Denhardt's 5' side ofthe DNA-binding region ofthe gene and 560 bp from solution, 5 x SSPE, 1% NaDodSO4, 10% (wt/vol) deXtran the putative initiation start site for transcription (20). The sulfate, heparin (25 units/ml), 0.25 mM ATP, and denatured human coagulation factor VIII DNA probe was a 5.4-kilobase salmon sperm DNA (100 ,g/ml). (1 x Denhardt's solution = pair (kb) genomic fragment from exon 26 of this gene, kindly 0.02% polyvinylpyrrolidone/0.02% Ficoll/0.02% bovine se- provided by Stylios Antonarakis (Johns Hopkins Univer- rum albumin; 1 x SSPE = 0.18 M NaCl/10 mM sodium sity). All DNA probes were radiolabeled to a specific activity phosphate, pH 7.4/1 mM EDTA.) Hybridization was per- of 109 cpm/,tg of DNA with [a-32P]dCTP (New England formed by adding the radiolabeled cDNA probe in the same Nuclear) by a random-priming method (22). prehybridization solution to a final concentration of 106 DNA Restriction Analysis. Genomic DNA was isolated cpm/ml and incubating at 42°C for 24-48 hr. The membranes from peripheral leukocytes as described (23) and modified by were washed successively in (i) 2 x SSC/0.5% NaDodSO4 our laboratory (24). For extraction of DNA from skin for two 15-min periods at 25°C, (ii) 2 x SSC/0.1% NaDodSO4

DNA-Binding Steroid- Binding Domain Domain I 5, - 31 I ---FAAAAA ATG I i I I Stop i

Eeo Hind Eco ECo ml hAR-3 I U hAR-l RI hAR-2 RI (575 bp) (715bp) (490 bp)

1 o 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Kb FIG. 2. Diagram of the human androgen receptor cDNA and the three fragments used as radiolabeled hybridization probes on Southern blots ofgenomic DNA. The human androgen receptor from its 5' initiation site (ATG) to its 3' stop codon is encoded by -2800 nucleotides. In addition, =500 bp of noncoding sequence extends from the stop codon to the beginning of the poly(A) tail. The positions of the DNA- (stippled bar) and steroid- (solid bar) binding domains are shown. The three cDNA probes used for hybridization and their respective positions within the cDNA structure are designated as hAR-1, hAR-2, and hAR-3. Downloaded by guest on September 28, 2021 Genetics: Brown et al. Proc. Nati. Acad. Sci. USA 85 (1988) 8153 for three 10-min periods at 250C, and (iii) 0.1 x SSC/l.Oo fragments were absent. As a control, the radiolabeled hAR-1 NaDodSO4 for four 30-min periods at 68-70'C. (1 x SSC = cDNA probe was removed and the same blots were rehybrid- 0.15 M NaCI/0.015 M sodium citrate, pH 7.0.) The mem- ized with a radiolabeled 5.4-kb genomic DNA probe for the branes were exposed to Kodak XAR-5 film with intensifying human factor VIII gene. This revealed the presence in all six screens at - 70'C for 3-10 days. Blots to be rehybridized with affected subjects of the expected restriction fragments for a second probe were first incubated three times with boiling EcoRI of 5.4 kb and forBamHI of 3.4 kb and 2.1 kb (data not 0.1 x SSC/0.5% NaDodSO4 and shaking to remove the shown). original hybridization probe. To ensure complete removal of Further documentation for a partial androgen receptor the probe, the blots were then exposed to XAR-5 film for 48 gene deletion was provided by DNA restriction fragment hr before rehybridization as described above. patterns of subject II-3 and other members of her family (Fig. 1). DNA digested with EcoRI and hybridized with radiola- RESULTS beled hAR-1 revealed the presence of two restriction frag- ments of 9.4 and 2.4 kb in the mother (1-2) and two normal Genomic DNA was extracted from cultured genital skin female siblings, 11-2 and II-9 (Fig. 4 Top), a pattern similar to fibroblasts or leukocytes of 10 normal subjects and 6 subjects that shown for three unrelated controls. Subject 11-3 and her with the receptor (-) form of complete AIS. DNA was affected nonidentical twin sibling (11-4) showed no evidence digested with the restriction endonuclease EcoRI or BamHI of a 2.4-kb fragment. Rehybridization of this same DNA blot and analyzed by Southern blotting techniques and by hybrid- with radiolabeled hAR-2 cDNA as probe (Fig. 4 Middle) ization with the hAR-1 probe. As shown in Fig. 3 Upper, two identified 1.2- and 0.7-kb restriction fragments in the three restriction fragments of9.4 kb and 2.4 kb were observed after controls, in the mother (1-2), and in the two unaffected female EcoRI digestion in 5 subjects (lanes A-E) with complete AIS siblings (II-2 and 11-9). However, the 1.2- and 0.7-kb restric- and also observed in 10 normal male and female subjects tion fragments were absent in 11-3 and II-4. By contrast, (data not shown). However, subject II-3 showed only the rehybridization of this same DNA blot with radiolabeled 9.4-kb fragment, suggesting that part of the androgen recep- hAR-3 cDNA probe revealed the presence of a restriction tor gene was missing. Further evidence for a partial deletion fragment of -23 kb for all subjects tested, including the two of the androgen receptor gene in subject II-3 was apparent with complete AIS (Fig. 4 Bottom). A control hybridization after DNA digestion with BamHI (Fig. 3 Lower). In 5 affected with the factor VIII probe confirmed that similar amounts of subjects (lanes A-E) and control subjects, the hAR-1 cDNA DNA were applied to each lane (data not shown). However, probe hybridized to restriction fragments of7.0, 6.0, 5.4, and the detection of the heterozygous carriers (e.g., subject 1-2) 4.4 kb. However, in subject 11-3 the 7.0- and 6.0-kb restriction Controls AIS A 11-3 B C D E Kb-2 I- 2 Subjects Z -2 -3 -9 y y e Kb Kb _4 9.4 - _ 0O.on ~~~~~A 9.4- **dUI'_ 1*,, le .4040 0W@*UII,

2.4 - 40 0

1.2- 0

2.4 - 40 MO ._I 40O __ 7.0 0.7- _ 30 -4, _ 0 W up _WW _ 6.0 - 4_ 5.4 23- ___ 4.4 - 0 -

FIG. 3. Southern blots of genomic DNA from genital skin FIG. 4. Southern blots of DNA from genital skin fibroblasts or fibroblasts of six unrelated subjects with the receptor (-) form of leukocytes of five members of the pedigree shown in Fig. 3 with complete AIS. DNA (8 ,ug) was digested with EcoRI (Upper) or complete AIS and of three control subjects (two females, one male). BamHI (Lower) and analyzed for hybridization with 32P-labeled DNA (10 Jg) was digested with EcoRI and analyzed by hybridization hAR-1 cDNA. with 32P-labeled hAR-1 (Top), hAR-2 (Middle), and hAR-3 (Bottom). Downloaded by guest on September 28, 2021 8154 Genetics: Brown et al. Proc. Natl. Acad. Sci. USA 85 (1988)

of the androgen receptor gene deletion in this family will Xq13. It has been shown that the androgen receptor struc- require a more definitive assessment ofgene dosage than that tural gene indeed occupies this X chromosome locus (20). shown in Fig. 4. Studies by Wieacker et al. (29) have also presented evidence The three androgen receptor cDNAs were used to probe for close linkage between the androgen receptor locus and DNA fragments generated by BamHI digestion in members DXS1 segment. of the family with complete AIS and in control subjects (Fig. The androgen receptor belongs to the subfamily of steroid 5). The radiolabeled hAR-1 cDNA probe hybridized with hormone receptors within a larger family of chromatin- restriction fragments of 7.0, 6.0, 5.4, and 4.4 kb in normal binding proteins that are likely to have evolved from a subjects, the mother (1-2), and two unaffected siblings (II-2 common ancestral gene (for review, see ref. 30). The steroid and 11-9) (Fig. 5 Top). However, 7.0- and 6.0-kb restriction receptor genes contain an amino-terminal region of variable fragments were absent in the two affected siblings (II-3 and length with a proposed role in transcriptional activation, a II-4). Radiolabeled hAR-2 cDNA hybridized with a single highly conserved central region rich in cysteine residues 2.0-kb restriction fragment in DNA from normal subjects, the required of the zinc-binding finger structure that binds DNA, mother, and two unaffected siblings, but not from the two and a carboxyl-terminal region where ligand binding occurs. affected subjects (Fig. 5 Middle). Rehybridization of this The cloning ofcDNAs for the human androgen receptor gene DNA blot with radiolabeled hAR-3 cDNA probe showed the (19, 20) provides the molecular tools to correlate structure presence of an -27-kb restriction fragment in all of the and function and, specifically, to identify and relate the subjects examined (Fig. 5 Bottom). natural mutations occurring in AIS to the physiologic prop- erties of androgen receptors. DISCUSSION In the present work, we elected to study patients with complete AIS who had undetectable androgen receptors by Complete unresponsiveness to androgens is known as AIS in ligand binding assays (31), in the expectation that some of humans and as the testicular feminization mutation in mice them might present a deletion of the androgen receptor gene. (25, 26) and rats (27, 28). Linkage to the X chromosome was About 5-10% of mutations found in genetic disorders are due first suggested in rats by analysis of sex ratios (27, 28), to gene deletions (32-35). Assuming a 5% frequency, screen- whereas the testicular feminization mutation in mice was ing of 14 independent mutations of the androgen receptor mapped to a linkage group, including tabby and blotchy, that gene should result in greater than a 50% chance of finding a occupies the distal half of the murine X chromosome (25, 26). gene deletion. Our group of patients included one affected Migeon et al. (5), by producing a series of testicular femini- subject from each of six unrelated families. One of them zation mutation mouse-human cell hybrids and testing their showed a definite deletion when using two endonucleases, expression of androgen receptor binding activity, demon- EcoRI and BamHI. This finding demonstrates further genetic strated that the androgen receptor gene or the gene locus of heterogeneity in AIS; some patients with the complete a factor controlling androgen receptor gene expression was receptor (-) form have a deletion of the androgen receptor located on the human X chromosome between Xpl1 and gene, whereas others have no detectable deletion when assessed by identical techniques. In the second group, the gene may not be expressed because ofa point mutation or the Controls mutant gene may express a receptor protein that is structur- ally modified so as to be unable to bind the androgen ligand. IE 2 - 3 - 4 - 9 Additional DNA restriction analysis was carried out in the patient with identifiable gene deletion and in members of her family. The use ofthree cDNA probes that spanned the entire receptor gene demonstrated that the deletion was partial but 6.0- affected the entire steroid-binding domain. This was demon- 5.4-- .A. strated by the absence of several restriction fragments that hybridized with hAR-1 cDNA, including the 5' portion ofthis domain, by the complete absence of the normal restriction 4.4~ I 0 4 fragments that hybridized with hAR-2 cDNA, including the 3' portion of the steroid-binding domain, but by a normal restriction fragment pattern with the hAR-3 cDNA probe, including a portion of the 5' amino-terminal domain (Fig. 2) of the androgen receptor. A dizygotic affected sister, two 2.0- unaffected sisters, and the mother confirmed the relation of 40 41 ol AIS and restriction fragments observed with the two endo- nucleases and the three cDNA probes used in the study. In summary, we have identified a partial deletion of the human androgen receptor gene in a subject with complete AIS. The fact that cultured genital skin fibroblasts from this subject have undetectable androgen receptor binding activity is correlated with an apparent deletion in the 3' region of the 27 - _ ow gene that encodes the carboxyl terminus of the receptor 4'm low- 4. protein responsible for androgen binding. Initial findings -.0.",t. ... from other subjects with the receptor (-) form of complete AIS suggest that gross deletions within the steroid-binding domain of the receptor do not account for their target cell FIG. 5. Southern blots of DNA from genital skin fibroblasts or insensitivity and hence these mutations may form a hetero- leukocytes of five members of the pedigree shown in Fig. 3 with geneous group of molecular lesions. complete AIS and of three control subjects (two females, one male). DNA (10 Ag) was digested with BamHI and analyzed by hybridiza- We thank Ms. Helen Linhard for the dedicated and expert tion with 32P-labeled hAR-1 (Top), hAR-2 (Middle), and hAR-3 technical assistance in the preparation of the DNA samples and their (Bottom). analysis by DNA hybridization and Ms. Shirley Ho for caring and Downloaded by guest on September 28, 2021 Genetics: Brown et al. Proc. Natl. Acad. Sci. USA 85 (1988) 8155

maintenance of the cell cultures. We are grateful to Drs. Gary 15. Brown, T. R., Maes, M., Rothwell, S. W. & Migeon, C. J. Berkovitz and John Rock for their assistance in obtaining the various (1982) J. Clin. Endocrinol. Metab. 55, 61-69. blood and skin specimens from the patients with androgen insensi- 16. Rivarola, M. A., Saez, J. M., Meyer, W. J., III, Kenny, F. M. tivity and to Dr. Patricia Donohoue for helpful discussions while & Migeon, C. J. (1967)J. Clin. Endocrinol. Metab. 27, 371-378. conducting these experiments. This work was supported by Grants 17. Kaufman, M., Pinsky, L., Baird, P. A. & McGillivray, B. C. HD-19536 and DK-00180 from the National Institutes of Health; (1979) Am. J. Med. Genet. 4, 402-411. Biomedical Research Support Grant RR-5378; and Grants HD-16910 18. Hughes, I. A. & Evans, B. R. (1986) J. Clin. Endocrinol. and HD-04466 from the National Institutes of Health, and P-30-HD- Metab. 63, 309-315. 18968 from the National Institute of Child Health and Human 19. Chang, C., Kokontis, J. & Liao, S. (1988) Science 240, 324-326. Development Center for Population Research. 20. Lubahn, D. B., Joseph, D. R., Sullivan, P. M., Willard, H. F., French, F. S. & Wilson, E. M. (1988) Science 240, 327-330. 1. Fichman, K., Migeon, B. & Migeon, C. J. (1980) Adv. Hum. 21. Tremblay, R. R., Foley, T. P., Jr., Corvol, P., Park, I.-J., Genet. 10, 331-377. Kowarski, A., Blizzard, R. M., Jones, H. W., Jr., & Migeon, 2. Brown, T. R. (1987) in Sexual Seminars in C. J. (1972) Acta Endocrinol. (Copenhagen) 70, 331-341. Differentiation, 22. Feinberg, A. P. & Vogelstein, B. (1984) Anal. Biochem. 137, Reproductive Biology, ed. Rock, J. A. (Thieme, New York), 266-277. Vol. 5, pp. 243-258. 23. Bell, G. I., Karam, J. M. & Rutter, W. J. (1981) Proc. Natl. 3. Brown, T. R. & Migeon, C. J. (1987) in Hormone Resistance Acad. Sci. USA 78, 5759-5763. and Other Endocrine Paradoxes, eds. Cohen, M. P. & Foa, 24. Donohoue, P. A., Van Dop, C., McLean, R. H., White, P. C., P. P. (Springer, New York), Vol. 1, pp. 157-203. Jospe, N. & Migeon, C. J. (1986) J. Clin. Endocrinol. Metab. 4. Meyer, W. J., III, Migeon, B. R. & Migeon, C. J. (1975) Proc. 62, 995-1002. Nati. Acad. Sci. USA 72, 1469-1472. 25. Lyon, M. F. & Hawkes, S. G. (1970) Nature (London) 227, 5. Migeon, B. R., Brown, T. R., Axelman, J. & Migeon, C. J. 1217-1219. (1981) Proc. Natl. Acad. Sci. USA 78, 1469-1472. 26. Ohno, S. & Lyon, M. F. (1970) Clin. Genet. 1, 121-129. 6. Keenan, B. S., Meyer, W. J., III, Hadjian, A. J., Jones, 27. Stanley, A. J. & Gumbreck, L. G. (1964) Proc. Endocrine Soc. H. W., Jr., & Migeon, C. J. (1974) J. Clin. Endocrinol. Metab. 46, 40 (abstr.). 38, 1143-1146. 28. Stanley, A. J., Gumbreck, L. G., Allsion, J. E. & Easley, 7. Griffin, J. E., Punyashthiti, K. & Wilson, J. D. (1976) J. Clin. R. B. (1973) Rec. Prog. Horm. Res. 29, 43-64. Invest. 57, 1342-1351. 29. Wieacker, P., Griffin, J. E., Wienker, T., Lopez, J. M., Wil- 8. Kaufman, M., Straisfeld, C. & Pinsky, L. (1976) J. Clin. Invest. son, J. D. & Breckwoldt, M. (1987) Hum. Genet. 76, 248-252. 58, 345-350. 30. Evans, R. M. (1988) Science 240, 889-895. 9. Migeon, C. J., Brown, T. R. & Fichman, K. (1981) in The 31. Brown, T. R. & Migeon, C. J. (1981) Mol. Cell. Biochem. 36, Child, ed. Josso, N. (Karger, Basel), Vol. 8, pp. 171- 3-22. 202. 32. Monaco, A. P., Bertelsen, C. J., Middlesworth, W., Colletti, 10. Amrhein, J. A., Meyer, W. J., III, Jones, H. W., Jr., & C. A., Aldridge, J., Fi~hbeck, K. H., Bartlett, R., Pericak- Migeon, C. J. (1976) Proc. Natl. Acad. Sci. USA 73, 891-894. Vance, M. A., Roses, A. D. & Kunkel, I. M. (1985) Nature 11. Aiman, J., Griffin, J. E., Gazak, J. M., Wilson, J. D. & (London) 316, 842-845. MacDonald, P. C. (1979) N. Engl. J. Med. 300, 223-227. 33. Antonarakis, S. E., Waber, P. G., Kittur, S. D., Patel, A. S., 12. Migeon, C. J., Brown, T. R., Lanes, R., Palacios, A., Am- Kazazian, H. H., Jr., Mellis, M. A., Counts, R. B., Stamato- rhein, J. A. & Schoen, E. J. (1984) J. Clin. Endocrinol. Metab. yannopoulos, G., Bowie, E. J. W., Fass, D. N., Pittman, 59, 672-678. D. D., Wozney, J. M. & Toole, J. J. (1985) N. Engl. J. Med. 13. Griffin, J. E. & Wilson, J. D. (1986) in Steroid Hormone 313, 842-848. Resistance: Mechanisms and Clinical Aspects, eds. Chrousos, 34. Giannelli, F., Choo, K. H., Rees, D. J. G., Boyd, Y., Rizza, G. P., Loriaux, D. L. & Lipsett, M. B. (Plenum, New York), C. R. & Brownleee, G. G. (1983) Nature (London) 303, 181- Vol. 196, pp. 257-268. 182. 14. Pinsky, L., Kaufman, M., Gil-Esteban, C. & Sumbulian, D. 35. Hobbs, H. H., Brown, M. S., Russell, D. W., Davignon, J. & (1983) Can. J. Biochem. Cell Biol. 71, 770-778. Goldstein, J. L. (1987) N. Engl. J. Med. 317, 734-737. Downloaded by guest on September 28, 2021