Hum Genet (1992) 90:311-312
human .. genetics 9 Springer-Verlag 1992
Point mutation in the steroid-binding domain of the androgen receptor gene in a family with complete androgen insensitivity syndrome (CAIS) Sibylle Jakubiczka 1, Edmond A. Werder 2, Peter Wieacker 1 lInstitut ftir Humangenetik, Medizinische Hochschule, Konstanty-Gutschow-Strasse 8, W-3000 Hannover 72, Federal Republic of Germany 2Ostschweizerisches Kinderspital, Claudiusstrasse 6, CH-9006 St. Gallen, Switzerland Received: 24 June 1992 / Revised: 22 July 1992
Abstract. An exonic single nucleotide substitution in the human androgen receptor gene ( h A R ) could be detected in an Italian family with two children affected by complete androgen insensitivity syndrome (CAIS), also called testicular feminization. This mutation leads to a guanine to adenine transition in exon 5, changing the sense of the codon from methionine ( A T G ) to valine ( G T G ) . As this mutation abolishes a N c o I restriction site, a rapid test for the mutation can be p e r f o r m e d by digestion of the polymerase chain reaction products with this enzyme. Previous results of indirect gene diagnosis in this family could be confirmed by this method.
Introduction Inadequate androgen action in genetic and gonadal males causes several androgen-resistance syndromes. The phenotypes vary from w o m e n with normal female external genitalia through patients with genital ambiguity to men with normal male genitalia but infertility. A t the functional level, deficient, defective, and quantitatively and qualitatively normal androgen receptors can be distinguished. The cloning of the c D N A for the h u m a n androgen receptor gene (Chang et al. 1988; Lubahn et al. 1988; Tilley et ah 1989; T r a p m a n et ah 1988) has made possible the molecular elucidation of these disorders. In an Italian family with complete androgen insensitivity syndrome (CAIS) we sought the molecular defect and found a point mutation in the steroid-binding domain.
Case report The pedigree of the family is given in Fig. 1. Cases II-1 and II-3 show the typical clinical and endocrinological features of CAIS. Correspondence to: P. Wieacker
Fig. 1. Pedigree of the family. Q and q, alleles of the DXS1/TaqI polymorphism. NcoI digestion of the PCR products from exon 5. M, Marker; K, control
They presented with normal external female genitalia, inguinal herniae and palpable gonads. In the proband (II-1), gonadal biopsy showed testicular tissue. At the age of 1.3 years, injection of human chorion gonadotropin (2500 IU) induced a rise of plasma testosterone from 20 to 331ng/100 ml within 3 days. At the age of 13.7 years, her breast development was at Tanner stage 4, pubic hair was scant (stage 3). At ultrasonography, vaginal length was estimated to be 5 cm and no uterus could be detected. Androgen receptor studies in skin fibroblasts could not be performed. The karyotypes of II-1 and II-3 are 46,XY, respectively.
Materials and methods DNA preparation from EDTA blood samples, restriction enzyme digestion, and Southern blot analysis using nick-labeled hybridization probe p8 (DXS1) were performed as previously described (Wieacker et ah 1987). DNA sequences were amplified by the polymerase chain reaction (PCR) according to Saiki et ah (1988) using the intronic primers of Lubahn et ah (1989) for exons 2-7, and the primers of Trifiro et ah (1991) for exon 8. Routinely, 35 reaction cycles were run in an Intelligent Heating Block (Biometra): 60 s at 93~ 60 s at the annealing temperature (exon 2: 62~ exon
312 c e p t o r s ( C h a n g et al. 1988). In exon 2 - 8 , this m u t a t i o n is the o n l y D N A a l t e r a t i o n that we c o u l d detect. W e cannot e x c l u d e m u t a t i o n s in e x o n 1 a n d / o r splicing defects in the o t h e r exons, a l t h o u g h the i m m e d i a t e e x o n / i n t r o n j u n c t i o n s have be f o u n d to b e n o r m a l . M e t h i o n i n e a n d valine are functionally similar a m i n o acids. N e v e r t h e l e s s , the l o c a l i z a t i o n of the m u t a t i o n is strong e v i d e n c e for this i n t e r c h a n g e b e i n g the m o l e c u l a r d e f e c t o p e r a t i v e in this family. This is s u p p o r t e d by a n o t h e r valine to methionine i n t e r c h a n g e in c o d o n 866, c a u s e d b y a G T G to A T G t r a n s i t i o n , a s s o c i a t e d with a d e c r e a s e in b i n d i n g affinity for a n d r o g e n ( L u b a h n et al. 1989). Fig. 2. Guanine to adenine transition in exon 5 (II-1)
3, 4, 6: 58~ exon 5 68~ exon 7: 60~ exon 8: 50~ 60 s at 72~ Double-stranded PCR products were separated by three ultrafiltration steps through Centricon-columns (Amicon); 1/4 of the product was then applied to 18 cycles of asymmetric PCR using only one of the primers (D6rk et al. 1991): 50pM primer, 200gM dNTP and 2.5 U Taq polymerase in 100 jal suppliers' buffer (Amersham). Single-stranded PCR products were then separated by two more ultrafiltration steps, and finally sequenced using an Sequenase Sequencing Kit Version 2.0 (USB) without denaturation of the probe.
Results and discussion D i r e c t s e q u e n c i n g of P C R - a m p l i f i e d exons 2 - 8 r e v e a l e d a n u c l e o t i d e s u b s t i t u t i o n in the h A R g e n e in o n e of the a f f e c t e d c h i l d r e n ( I L l ) . T h e m u t a t i o n , a G to A transition o c c u r r e d at c o d o n 749 in e x o n 5 o f the g e n e , l e a d i n g to a m e t h i o n i n e to valine interchange (Fig. 2). T h e m o t h e r ( I - l ) a n d t h e sister (II-2) are h e t e r o z y g o u s for the m u t a tion, w h e r e a s the o t h e r a f f e c t e d child of the family (II-3) is also h e m i z y g o u s . This can b e shown b y direct s e q u e n c ing o r m o r e r a p i d l y by NcoI d i g e s t i o n o f the d o u b l e s t r a n d e d P C R p r o d u c t s , as t h e m u t a t i o n a b o l i s h e s a restriction site for this e n z y m e . A s e x p e c t e d , we f o u n d , in II-1 a n d II-3, o n l y the uncut P C R p r o d u c t of 285 b p in length, w h e r e a s the P C R p r o d u c t o f the f a t h e r ( I - l ) shows t h e n o r m a l b a n d s of 162 a n d 123 bp. T h e m o t h e r (I-2) a n d the sister (II-2) a r e carriers o f the m u t a t i o n , as t h e y show b o t h the m u t a n t a n d u n a f f e c t e d alleles. This test c o n f i r m s the results of the l i n k a g e analysis with the DXS1/TaqI p o l y m o r p h i s m ( W i e a c k e r et al, 1987). In this family, the gene for a n d r o g e n insensitivity is in p h a s e with allele Q: I-2 is h e t e r o z y g o u s ( Q q ) , I1-1 a n d II-3 are h e m i z y g o u s for allele Q , w h e r e a s II-2 has i n h e r i t e d q f r o m h e r f a t h e r and Q f r o m the m o t h e r . T h e m u t a t i o n d e s c r i b e d h e r e is l o c a l i z e d in the s t e r o i d - b i n d i n g d o m a i n of the r e c e p t o r in a r e g i o n c o n s e r v e d a m o n g s t e r o i d re-
Acknowledgements. We thank J. Schmidtke for helpful discussions. This work was supported by the Deutsche Forschungsgemeinschaft through a grant to P.W. (Wie 792/2-2).
References Chang C, Kokontis J, Liao S (1988) Structural analysis of complementary DNA and amino acid sequences of human and rat androgen receptors. Proc Natl Acad Sci USA 85:7211-7215 D6rk T, Wullbrand U, Richter T, Neumann T, Wolfes H, Wulf B, Maass G, Tfimmler B (1991) Cystic fibrosis with three mutations in the cystic fibrosis transmembrane conductance regulator gene. Hum Genet 87:441-446 Lubahn DB, Joseph DR, Sullivan PM, Willard HF, French FS. Wilson EM (1988) Cloning of human androgen receptor complementary DNA and localization to the X-chromosome. Science 240 : 237-330 Lubahn DB, Brown TR, Simental JA, Higgs HN, Migeon CJ, Wilson EM, French FS (1989) Sequence of the intron/exon junctions of the coding region of the human androgen receptor gene and identification of a point mutation in a family with complete androgen insensitivity. Proc Natl Acad Sci USA 86: 9534-9538 Saiki RK, Gelfand DH, 8toffel S, Scharf J, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988) Primer-directed enzymatic amplification of DNA with thermostable DNA-polymerase. Science 239:487-491 Tilley WD, Marcelli M, Wilson JD, McPhaul MJ (1989) Characterization and expression of a cDNA encoding the human androgen receptor. Proc Natl Acad Sci USA 86:327-331 Trapman J, Klaassen P, Kuiper GGJM, Korput JAGM van der, Faber PW, Rooij HCJ van, Kessel AG van, Voorhorst MM, Mulder E, Brinkmann AO (1988) Cloning, structure and expression of a cDNA encoding the human androgen receptor. Biochem Biophys Res Communun 153 : 241-248 Trifiro M, Prior LP, Sabbaghian N, Pinsky L, Kaufmann M, Nylen EG, Belsham DD, Greenberg CR, Wrogemann K (1991) Amber mutation creates a diagnostic Mad site in the androgen receptor gene of a family with complete androgen insensitivity. Am J Hum Genet 40 : 493-499 Wieacker P, Griffin JE, Wienker T, Lopez JM, Wilson JD, Breckwoldt M (1987) Linkage analysis with RFLPs in families with androgen resistance syndromes: evidence for close linkage between the androgen receptor locus and the DXS1 segment. Hum Genet 76 : 248-252
human .. genetics 9 Springer-Verlag 1992
Point mutation in the steroid-binding domain of the androgen receptor gene in a family with complete androgen insensitivity syndrome (CAIS) Sibylle Jakubiczka 1, Edmond A. Werder 2, Peter Wieacker 1 lInstitut ftir Humangenetik, Medizinische Hochschule, Konstanty-Gutschow-Strasse 8, W-3000 Hannover 72, Federal Republic of Germany 2Ostschweizerisches Kinderspital, Claudiusstrasse 6, CH-9006 St. Gallen, Switzerland Received: 24 June 1992 / Revised: 22 July 1992
Abstract. An exonic single nucleotide substitution in the human androgen receptor gene ( h A R ) could be detected in an Italian family with two children affected by complete androgen insensitivity syndrome (CAIS), also called testicular feminization. This mutation leads to a guanine to adenine transition in exon 5, changing the sense of the codon from methionine ( A T G ) to valine ( G T G ) . As this mutation abolishes a N c o I restriction site, a rapid test for the mutation can be p e r f o r m e d by digestion of the polymerase chain reaction products with this enzyme. Previous results of indirect gene diagnosis in this family could be confirmed by this method.
Introduction Inadequate androgen action in genetic and gonadal males causes several androgen-resistance syndromes. The phenotypes vary from w o m e n with normal female external genitalia through patients with genital ambiguity to men with normal male genitalia but infertility. A t the functional level, deficient, defective, and quantitatively and qualitatively normal androgen receptors can be distinguished. The cloning of the c D N A for the h u m a n androgen receptor gene (Chang et al. 1988; Lubahn et al. 1988; Tilley et ah 1989; T r a p m a n et ah 1988) has made possible the molecular elucidation of these disorders. In an Italian family with complete androgen insensitivity syndrome (CAIS) we sought the molecular defect and found a point mutation in the steroid-binding domain.
Case report The pedigree of the family is given in Fig. 1. Cases II-1 and II-3 show the typical clinical and endocrinological features of CAIS. Correspondence to: P. Wieacker
Fig. 1. Pedigree of the family. Q and q, alleles of the DXS1/TaqI polymorphism. NcoI digestion of the PCR products from exon 5. M, Marker; K, control
They presented with normal external female genitalia, inguinal herniae and palpable gonads. In the proband (II-1), gonadal biopsy showed testicular tissue. At the age of 1.3 years, injection of human chorion gonadotropin (2500 IU) induced a rise of plasma testosterone from 20 to 331ng/100 ml within 3 days. At the age of 13.7 years, her breast development was at Tanner stage 4, pubic hair was scant (stage 3). At ultrasonography, vaginal length was estimated to be 5 cm and no uterus could be detected. Androgen receptor studies in skin fibroblasts could not be performed. The karyotypes of II-1 and II-3 are 46,XY, respectively.
Materials and methods DNA preparation from EDTA blood samples, restriction enzyme digestion, and Southern blot analysis using nick-labeled hybridization probe p8 (DXS1) were performed as previously described (Wieacker et ah 1987). DNA sequences were amplified by the polymerase chain reaction (PCR) according to Saiki et ah (1988) using the intronic primers of Lubahn et ah (1989) for exons 2-7, and the primers of Trifiro et ah (1991) for exon 8. Routinely, 35 reaction cycles were run in an Intelligent Heating Block (Biometra): 60 s at 93~ 60 s at the annealing temperature (exon 2: 62~ exon
312 c e p t o r s ( C h a n g et al. 1988). In exon 2 - 8 , this m u t a t i o n is the o n l y D N A a l t e r a t i o n that we c o u l d detect. W e cannot e x c l u d e m u t a t i o n s in e x o n 1 a n d / o r splicing defects in the o t h e r exons, a l t h o u g h the i m m e d i a t e e x o n / i n t r o n j u n c t i o n s have be f o u n d to b e n o r m a l . M e t h i o n i n e a n d valine are functionally similar a m i n o acids. N e v e r t h e l e s s , the l o c a l i z a t i o n of the m u t a t i o n is strong e v i d e n c e for this i n t e r c h a n g e b e i n g the m o l e c u l a r d e f e c t o p e r a t i v e in this family. This is s u p p o r t e d by a n o t h e r valine to methionine i n t e r c h a n g e in c o d o n 866, c a u s e d b y a G T G to A T G t r a n s i t i o n , a s s o c i a t e d with a d e c r e a s e in b i n d i n g affinity for a n d r o g e n ( L u b a h n et al. 1989). Fig. 2. Guanine to adenine transition in exon 5 (II-1)
3, 4, 6: 58~ exon 5 68~ exon 7: 60~ exon 8: 50~ 60 s at 72~ Double-stranded PCR products were separated by three ultrafiltration steps through Centricon-columns (Amicon); 1/4 of the product was then applied to 18 cycles of asymmetric PCR using only one of the primers (D6rk et al. 1991): 50pM primer, 200gM dNTP and 2.5 U Taq polymerase in 100 jal suppliers' buffer (Amersham). Single-stranded PCR products were then separated by two more ultrafiltration steps, and finally sequenced using an Sequenase Sequencing Kit Version 2.0 (USB) without denaturation of the probe.
Results and discussion D i r e c t s e q u e n c i n g of P C R - a m p l i f i e d exons 2 - 8 r e v e a l e d a n u c l e o t i d e s u b s t i t u t i o n in the h A R g e n e in o n e of the a f f e c t e d c h i l d r e n ( I L l ) . T h e m u t a t i o n , a G to A transition o c c u r r e d at c o d o n 749 in e x o n 5 o f the g e n e , l e a d i n g to a m e t h i o n i n e to valine interchange (Fig. 2). T h e m o t h e r ( I - l ) a n d t h e sister (II-2) are h e t e r o z y g o u s for the m u t a tion, w h e r e a s the o t h e r a f f e c t e d child of the family (II-3) is also h e m i z y g o u s . This can b e shown b y direct s e q u e n c ing o r m o r e r a p i d l y by NcoI d i g e s t i o n o f the d o u b l e s t r a n d e d P C R p r o d u c t s , as t h e m u t a t i o n a b o l i s h e s a restriction site for this e n z y m e . A s e x p e c t e d , we f o u n d , in II-1 a n d II-3, o n l y the uncut P C R p r o d u c t of 285 b p in length, w h e r e a s the P C R p r o d u c t o f the f a t h e r ( I - l ) shows t h e n o r m a l b a n d s of 162 a n d 123 bp. T h e m o t h e r (I-2) a n d the sister (II-2) a r e carriers o f the m u t a t i o n , as t h e y show b o t h the m u t a n t a n d u n a f f e c t e d alleles. This test c o n f i r m s the results of the l i n k a g e analysis with the DXS1/TaqI p o l y m o r p h i s m ( W i e a c k e r et al, 1987). In this family, the gene for a n d r o g e n insensitivity is in p h a s e with allele Q: I-2 is h e t e r o z y g o u s ( Q q ) , I1-1 a n d II-3 are h e m i z y g o u s for allele Q , w h e r e a s II-2 has i n h e r i t e d q f r o m h e r f a t h e r and Q f r o m the m o t h e r . T h e m u t a t i o n d e s c r i b e d h e r e is l o c a l i z e d in the s t e r o i d - b i n d i n g d o m a i n of the r e c e p t o r in a r e g i o n c o n s e r v e d a m o n g s t e r o i d re-
Acknowledgements. We thank J. Schmidtke for helpful discussions. This work was supported by the Deutsche Forschungsgemeinschaft through a grant to P.W. (Wie 792/2-2).
References Chang C, Kokontis J, Liao S (1988) Structural analysis of complementary DNA and amino acid sequences of human and rat androgen receptors. Proc Natl Acad Sci USA 85:7211-7215 D6rk T, Wullbrand U, Richter T, Neumann T, Wolfes H, Wulf B, Maass G, Tfimmler B (1991) Cystic fibrosis with three mutations in the cystic fibrosis transmembrane conductance regulator gene. Hum Genet 87:441-446 Lubahn DB, Joseph DR, Sullivan PM, Willard HF, French FS. Wilson EM (1988) Cloning of human androgen receptor complementary DNA and localization to the X-chromosome. Science 240 : 237-330 Lubahn DB, Brown TR, Simental JA, Higgs HN, Migeon CJ, Wilson EM, French FS (1989) Sequence of the intron/exon junctions of the coding region of the human androgen receptor gene and identification of a point mutation in a family with complete androgen insensitivity. Proc Natl Acad Sci USA 86: 9534-9538 Saiki RK, Gelfand DH, 8toffel S, Scharf J, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988) Primer-directed enzymatic amplification of DNA with thermostable DNA-polymerase. Science 239:487-491 Tilley WD, Marcelli M, Wilson JD, McPhaul MJ (1989) Characterization and expression of a cDNA encoding the human androgen receptor. Proc Natl Acad Sci USA 86:327-331 Trapman J, Klaassen P, Kuiper GGJM, Korput JAGM van der, Faber PW, Rooij HCJ van, Kessel AG van, Voorhorst MM, Mulder E, Brinkmann AO (1988) Cloning, structure and expression of a cDNA encoding the human androgen receptor. Biochem Biophys Res Communun 153 : 241-248 Trifiro M, Prior LP, Sabbaghian N, Pinsky L, Kaufmann M, Nylen EG, Belsham DD, Greenberg CR, Wrogemann K (1991) Amber mutation creates a diagnostic Mad site in the androgen receptor gene of a family with complete androgen insensitivity. Am J Hum Genet 40 : 493-499 Wieacker P, Griffin JE, Wienker T, Lopez JM, Wilson JD, Breckwoldt M (1987) Linkage analysis with RFLPs in families with androgen resistance syndromes: evidence for close linkage between the androgen receptor locus and the DXS1 segment. Hum Genet 76 : 248-252
