17,20-lyase defects

.7. Steroid Biochem. Molec. Biol. Vol. 53, No. 1-6, pp. 153-157, 1995

Pergamon

17

0960-0760(95)00029-1

Copyright ~(~ 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0960-0760/95 $9.50 + 0.00

-Hydroxylase/17,20-1yase Defects Toshihiko Yanase

The Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka 812, Japan C o n g e n t i a l a d r e n a l h y p e r p l a s i a due to 170t-hydroxylase/17/20-1yase d e f i c i e n c y is c a u s e d by genetic d e f ects in th e gene e n c o d i n g P450c17 (CYP17). To date, 18 d i f f e r e n t m u t a t i o n s in 27 i n d i v i d u a l s h a v e b e e n i d e n t i f i e d a n d all o f t h e m a r e l o c a t e d in t he codi ng r e g i o n o f CYP17. S e v e r a l m u t a t i o n s h a v e b e e n r e c o n s t r u c t e d in h u m a n P450c17 cDNA a n d e x p r e s s e d in CO S cells to c h a r a c t e r i z e t he k in e tic p r o p e r t i e s o f 17at-hydroxylase a n d 17,20-1yase activities. T h e m o l e c u l a r bases o f cases clinically r e p o r t e d as 1 7 ~ - h y d r o x y l a s e d e f i c i e n c y h a v e t u r n e d o u t to r e s u l t f r o m c o m p l e t e or p a r t i a l c o m b i n e d deficiencies o f 17ot-hydroxylase/17,20-1yase. T h e e l u c i d a t i o n o f t he m o l e c u l a r bases g e n e r a l l y exp l a in s t h e p a t i e n t ' s clinical profiles i n c l u d i n g t h e sexual p h e n o t y p e o f t he e x t e r n a l genitalia. In o n e case i n i t i a l l y r e p o r t e d as i s o l a t e d 17,20-1yase deficiency, t he m o l e c u l a r basis was f o u n d to be p a r t i a l c o m b i n e d d e f i c i e n c y o f b o t h activities, s o m e w h a t d i s c o r d a n t w i t h t he p a t i e n t ' s clinical profile. H o w e v e r , t h e p a t i e n t was s u b s e q u e n t l y f o u n d to h a v e 1 7 a - h y d r o x y l a s e deficiency, suggesting i n v o l v e m e n t s o f a g e - d e p e n d e n t u n k n o w n f a c t o r s affecting P450c17 activity.

J. Steroid Biochem. Molec. Biol., Vol. 53, No. 1-6, pp. 153-157, 1995

INTRODUCTION

seem to be a partial form of this trait [6]. On the other hand, a third type of deficiency, isolated 17,20-1yase deficiency, is also described in the literature. This is much less common than 17a-hydroxylase deficiency and only 14 cases have been reported [6]. Reports on genetic analysis of each form of P450c17 defect have been accumulated. In this article, the molecular bases of all of the reported cases of P450c17 deficiency are briefly reviewed.

A relatively rare cause of congenital adrenal hyperplasia (CAH), "lTct-hydroxylase deficiency" can be characterized by a defect in either or both of the 17ct-hydroxylase and 17,20-1yase activities, based on the fact that a single polypeptide, P450c17 catalyzes two distinct reactions: 17~t-hydroxylation of pregnenolone or progesterone and the 17,20-1yase reaction of 17~-hydroxylated pregnenolone to yield the C19 steroid precursors of testosterone and estrogens [1,2]. T h e CYP17 gene encoding P450c17 [3, 4] is a single copy gene and located on chromosome 10q24-25 [5]. Deficiency of P450c17 normally present in both the gonads and adrenal cortex leads to impaired production of cortisol, androgens and estrogens, with accompanying overproduction of mineralocorticoids, in particular 11-deoxycorticosterone (DOC). Consequently, affected females (46XX) have hypertension and absence of sexual development producing primary amenorrhea, while affected males (46XY) are also hypertensive and have female external genitalia. Rarely, partial deficiency of this enzyme associated with genetic ambiguity have been reported. T o date, more than 120 cases of 17~-hydroxylase deficiency have been reported [6]. While most of them are clinically reported to have complete 17~-hydroxylase deficiency, about 20 cases

MOLECULAR B A S I S OF 17~-HYDROXYLASE AND 17,20-LYASE DEFICIENCY Patient CYP17 was analyzed by exonic sequencing following a conventional cloning method [3] or polymerase chain reaction (PCR) [7]. T o date, 18 different mutations in 27 individuals have been identified and all of them are located in the structural gene [3, 8-22 and T. Imai, unpublished observation]. Table 1 is a brief summary of the clinical profiles and molecular defects of these patients.

Complete combined 17~-hydroxylase and 17,20-lyase deficiencies Fourteen kinds of mutations which result in virtual loss of P450c17 activity have been identified in patients showing typical symptoms of 17~-hydroxylase deficiency (cases 1-24 in Table 1). Briefly, 6 are mutations causing non-sense mutations by themselves [8, 14] or as a result of alterations of the reading frame [11, 12, 19].

Proceedings of the I X International Congress on Hormonal Steroid, Dallas, Texas, U.S.A., 24-29 September 1994. 153

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Toshihiko Yanase

Table 1. Summary of clinical profiles and molecular defects in which the structure of C Y P 17 gene has been analyzed Case

Karyotype

Socialsex

Mutation

Reference

1 (ML) 2 (BD) 3 4 5 6 7 8 9 10 11 (JY) 12 (JK) 13 (DG) 14 (DA) 15 (DM) 16 17 18

A B C C D E E F G H I J K K K L M N

Family

Canadian Canadian Netherlander Netherlander Netherlander Netherlander Netherlander Netherlander Netherlander Netherlander Japanese Japanese Italian Italian Italian Guamanian Guamanian English

Race

46XY 46XY 46XY 46XY 46XX 46XX 46XX 46XY 46XY 46XY 46XX 46XX 46XY 46XX 46XX 46XY 46XY 46XX

F F F F F F F F F M F F F F F F F F

3 9 10 10 10 10 10 10 10 10 8 11 12 12 12 13 13 14

19

O

Thai

46XX

F

20 21 22 (VB)

P Q R

Japanese Japanese Caucasian

46XX 46XX 46XY

F F F

23 24 (WV) 25 (JG) 26 (DL)

S T U V

German Caucasian Japanese Canadian

46XX 46XX 46XX 46XY

F F F M

27 (IS)

W

Swiss

46XY

F

4 bp duplication (480) Same as case 1 Same as case 1 Same as case 1 Same as case 1 Same as case 1 Same as case 1 Same as case 1 Same as case 1 Same as case 1 Trp(17)-~End 7 bp duplication (120) Deletion and insertion (Exon 2-3) Same as case 13 Same as case 13 Ser(106)-~Pro Same as case 16 (1) Glu(194)-~End (2) Arg(239)~End Deletion of Asp(487)-Ser(488)-Phe(489) His(373)~Leu 2-bp (GC) deletion (300, 301) (1) Tyr(64)--*Ser (2) Duplication of Ile (112) Arg(440)~ His Gly(90)~Asp Deletion of Phe (53 or 54) (1) Arg(239)-~End (2) Pro(342)--*Thr (1) Gln(461)--*End (2) Arg(496)~Cys

15 16 19 17 18 20 21 22

Case 10 is a heterozygous for the mutation and thus may have a different mutation on the other CYP17 allele. All cases except 18, 22, 26 and 27 are homozygous for their respective mutations. Mutation in case 24 is based on unpublished observation by T. Imai.

Since the p r e m a t u r e stop c o d o n is located at the a m i n o t e r m i n a l side of the h e m e - b i n d i n g cysteine (442) i n P 4 5 0 c 1 7 , the p r e s e n c e of these m u t a t i o n s leads to a b s e n c e of a f u n c t i o n a l P 4 5 0 c 1 7 p r o t e i n i n the a d r e n a l cortex a n d gonads. All p a t i e n t s are h o m o z y g o u s for t h e i r respective m u t a t i o n s except one (case 18) h a v i n g two different stop c o d o n s as a c o m p o u n d heterozygote [14]. A 4 b p ( C A T C ) d u p l i c a t i o n at 480 e x t i n g u i s h e s P 4 5 0 c 1 7 activity because it alters the r e a d i n g frame, l e a d i n g to a c a r b o x y - t e r m i n a l s e q u e n c e that is c o m p l e t e l y different f r o m that of n o r m a l s e q u e n c e a n d i n - f r a m e stop c o d o n at 506 [3, 9, 10]. T h e 4 b p d u p l i c a t i o n is f o u n d in m e m b e r s of the M e n n o n i t e religious sect r e s i d i n g i n C a n a d a (cases 1 a n d 2) a n d i n d i v i d u a l s i n F r i e s l a n d in H o l l a n d (cases 3-10), m a k i n g this m u t a t i o n the m o s t w i d e l y d i s t r i b u t e d cause of P 4 5 0 c 1 7 deficiency [3, 9, 10]. A t h r e e a m i n o acid ( A s p - S e r - P h e n ) d e l e t i o n at 4 8 7 - 4 8 9 is also s h o w n to d e s t r o y d e t e c t a b l e P 4 5 0 c 1 7 activity p r o b a b l y d u e to a c o n f o r m a t i o n a l c h a n g e of P 4 5 0 c 1 7 p r o t e i n [15]. T h e s e two m u t a t i o n s clearly tell us that the c a r b o x y - t e r m i n a l r e g i o n is i m p o r t a n t for its P 4 5 0 c 1 7 activities.

I n t e r e s t i n g l y , 4 m u t a t i o n s are single a m i n o acid s u b s t i t u t i o n s w h i c h c o m p l e t e l y e x t i n g u i s h detectable P 4 5 0 c 1 7 activities [13, 16, 18 a n d T . I m a i , u n p u b lished observation]. F r o m the m o l e c u l a r m o d e l i n g of the h u m a n P 4 5 0 c 1 7 s e q u e n c e based o n the tertiary s t r u c t u r e of bacterial P 4 5 0 c a m , it has b e e n p r e d i c t e d that Ser ( 1 0 6 ) ~ Pro m a y affect s u b s t r a t e b i n d i n g [13]. Similarly, His (373) -~ L e u has b e e n p r e d i c t e d to affect the h e m e - b i n d i n g a n d it has b e e n actually d e m o n strated that m e m b r a n e p r e p a r a t i o n s f r o m E. coli cells e x p r e s s i n g the m u t a n t form, u n l i k e those e x p r e s s i n g wild type, show n o P 4 5 0 n m peak u p o n r e d u c t i o n a n d a d d i t i o n of c a r b o n m o n o x i d e [16]. T h e m u t a t i o n of Arg (440) ~ His p r o b a b l y d i s t u r b s the h e m e - b i n d i n g since A r g (440) m a y be one of crucial residues c o n s t i t u t i n g the h e i n e - b i n d i n g r e g i o n of P 4 5 0 c 1 7 [18]. I n case 22 in T a b l e 1, c o m p o u n d h e t e r o z y g o u s m u t a t i o n s were f o u n d . U p o n e x p r e s s i o n of these m u t a n t p r o t e i n s in E. coli, T y r (64) -~ Ser m u t a n t has 15% of the wild type 1 7 a - h y d r o x y l a s e activity, whereas the Ile (112) d u p l i c a t i o n showed no activity. I n spite of the increased levels of corticosterone a n d D O C , s u p p r e s s e d levels of a l d o s t e r o n e are o b s e r v e d in

Defects in P450c17

155

TCT(Ser) 64

t

TAT(Tyr)

2x

112 ATC(lie) ACA(Thr)

2×

.... I a 518bp d e l e t i o n ~ 342 t 480 ~ o r o4 ~ [ w i t h a 469 b p i n s e r t i o n ~ ...._, . . . . . . (~TTC)J ~ " ~ ~ bt~/ALr'ro) UA/U ""'~(A ' l ~ n"~ TAG~G ~ ~ - 496 Ubb,, ,rg) T G ~ O ~ ( ~ C ~ y - s~ ) " ~

/ ~ GGC(Gly) 90 ~ GAC(Asp)

TAG 194

300,301 CGC(Arg) 487 488 489 ~ J 440~ CAC(His)

Fig. 1. S c h e m a t i c representation o f the h u m a n CYP17 gene showing the position of m u t a t i o n s reported to date [3, 6, 8-22 and T. Imai, u n p u b l i s h e d observations]. The n u m b e r e d boxes represent the exons; the lines between t h e m represent the introns. The circle noted in exon 8 represents the cysteine (442) required for h e m e binding by P450c17.

most patients with 17~-hydroxylase deficiency. T h e suppression of aldosterone secretion f r o m the zona glomerulosa is attributed to the suppression of the renin-angiotensin system caused by sodium retention and volume expanding actions of markedly elevated D O C and corticosterone secretion from the zona fasciculata. However, some cases with 17~-hydroxylase deficiency have elevated aldosterone levels and the m e c h a n i s m remains unclear [6]. Cases 20 and 21 are such cases with exceptionally high concentrations of plasma aldosterone. However, since both patients were found to have defective P450c17 [16, 19] as observed in other cases with normal or suppressed levels of plasma aldosterone concentration, other m e c h anisms for elevated aldosterone levels should be considered.

Partial combined 17~-hydroxylase / 17,20-lyase deftciency Only two cases of partial combined deficiencies of both activities have been analyzed at the molecular level (cases 25 and 26). A Japanese female patient, J G has irregular menstruation, suggesting some degree of estrogen production. Sequence analysis revealed a homozygous deletion of the phenylalanine codon ( T T C ) at either amino acid 53 or 54 in exon 1 [20]. On the other hand, D L (case 26) is a 46XY newborn male with ambiguous external genitalia, suggesting some degree of testosterone production. T h e patient was found to be

a c o m p o u n d heterozygote, carrying two different m u tant alleles in the CYP17. One allele contains a stop codon ( T G A ) in place of arginine ( C G A ) at amino acid position 239 in exon 4, which makes the resultant protein truncated and nonfunctional. T h e second allele contains a missense mutation, namely a change from proline (CCA) to threonine (ACA) at amino acid position 342 in exon 6 [21]. Expression studies of the mutant c D N A constructs containing a change from proline to threonine at 342 or the phenylalanine deletion at either 53 or 54 in Cos 1 cells made it possible to estimate P450c17 activities of each individual. With respect to the requirements of 17,20-1yase activity and the sexual phenotype, D L having ambiguous genitalia has been found to have 20% of the normal level of 17,20-1yase activity. We do know that a 46XY individual (JK) (case 12), expected to have no P450c17 activity because of the homozygous presence of a p r e m a t u r e stop codon has female external genitalia. In addition, the father of this patient, a heterozygous carrier for the p r e m a t u r e stop codon and therefore expected to have 50% of normal P450c17 activity has apparently normal reproductive capacity. T h u s , the threshold 17,20-1yase activity necessary for changing the sexual phenotype of the external genitalia from female to ambiguous is 0 - 2 0 % , while that for changing from ambiguous to normal is 2 0 - 5 0 % [21]. In genetic females, the threshold activity of 17,20-1yase for menstruation seems to be lower than expected since only 5 % normal

156

Toshihiko Yanase

17,20-1yase activity leads to i r r e g u l a r m e n s t r u a t i o n in the p a t i e n t J G [20]. 1 7,20-lyase deficiency

Case I S (case 27 i n T a b l e 1) was o r i g i n a l l y r e p o r t e d as b e i n g isolated 17,20-1yase deficiency w h e n he was 15 years old [23]. T h i s p a t i e n t was f o u n d to be a c o m p o u n d h e t e r o z y g o t e h a v i n g two different m u t a t i o n s in the C Y P 1 7 gene. O n e allele c o n t a i n s a m i s s e n s e m u t a t i o n f r o m a r g i n i n e ( C G C ) to cysteine ( T G C ) at 496 while the second allele has a stop c o d o n ( T A G ) i n place of g l u t a m i n e ( C A G ) at 461. E x p r e s s i o n studies to d e t e r m i n e the effect of each m u t a t i o n o n the e n z y m a t i c p r o p e r t i e s of P 4 5 0 c 1 7 revealed that in this i n d i v i d u a l , b o t h 17~t-hydroxylase a n d 17,20-1yase activities were d r a m a t i c a l l y r e d u c e d r a t h e r t h a n there o n l y b e i n g a r e d u c t i o n i n 17,20-1yase, as initially r e p o r t e d [23]. H o w e v e r , s u b s e q u e n t e n d o c r i n o l o g i c a l e x a m i n a t i o n s of this p a t i e n t w h e n 25 years old have s h o w n the a b s e n c e of b o t h 1 7 ~ - h y d r o x y l a s e a n d 17,20-1yase activities [24]. T h i s fact suggests the i n v o l v e m e n t of a g e - d e p e n d e n t u n k n o w n factors affecting P 4 5 0 c 1 7 activity. T h e elect r o n t r a n s f e r s y s t e m m a y be one of such factors since the relative activity ratio of 17,20-1yase/17~-hydroxylase is r e p o r t e d to increase several fold b y elevating the ratio of either N A D P H - c y t o c h r o m e P 4 5 0 - r e d u c t a s e (Red) [25] or c y t o c h r o m e b5 (b5) [26]. Differences in tissue b5 or R e d c o n c e n t r a t i o n have b e e n suggested to be f u n c t i o n a l l y associated with differences in 17,20lyase activity i n s t e r o i d o g e n i c tissues [25, 27, 28]. CONCLUSIONS I n the 27 p a t i e n t s s t u d i e d b i o c h e m i c a l l y to date, 18 different m u t a t i o n s are f o u n d s u g g e s t i n g that m o s t of the r e p o r t e d cases of 1 7 ~ - h y d r o x y l a s e deficiency arise f r o m r a n d o m alterations i n the s t r u c t u r e of the C Y P 1 7 gene. A n e x c e p t i o n to this is the defect b e i n g f o u n d in 10 p a t i e n t s that p r o b a b l y reflects a " F o u n d e r effect" w h i c h arose in the F r i e s l a n d r e g i o n of H o l l a n d . I n m o s t cases of 1 7 ~ - h y d r o x y l a s e deficiency, the m o l e c u l a r basis of the disease clearly defines the clinical s y m p toms. H o w e v e r , the rare case w h i c h s h o w e d a clinical c o n v e r s i o n f r o m 17,20-1yase deficiency to 1 7 ~ - h y droxylase deficiency with a g i n g suggests that m o l e c u l a r basis does n o t always e x p l a i n the clinical profiles a n d the necessity to c o n s i d e r o t h e r genetic or n o n g e n e t i c factors m o d u l a t i n g P 4 5 0 c 1 7 activity. A precise explan a t i o n of w h y each m u t a t i o n affects the P 4 5 0 c 1 7 activities r e m a i n s u n c l e a r except in cases with p r e m a t u r e stop c o d o n p o s i t i o n e d at the a m i n o t e r m i n a l side o f P 4 5 0 c 1 7 h e m e - b i n d i n g cysteine (442). T h e m e c h a n i s m will o n l y b e c o m e a p p a r e n t w h e n the t e r t i a r y s t r u c t u r e of h u m a n P 4 5 0 c 1 7 is d e t e r m i n e d . T h e r e c e n t d e v e l o p m e n t of the bacterial system for b o t h the e x p r e s s i o n a n d p u r i f i c a t i o n o f h u m a n P 4 5 0 c 1 7 [17] e n h a n c e s the p o s s i b i l i t y of o b t a i n i n g this structure.

Acknowledgements--The author appreciates greatly the important

contribution of Drs M. R. Waterman, E. R. Simpson, M. Kagimoto, K. Kagimoto, J. S. D. Winter, N. Matsui, A. Shibata, S. Suzuki, K. Hashiba, J. R. Pratt, M. Zachmann, R. Ahlgren, A. Biason, F. Mantero and T. Imai. These studies were supported in part by Grant FY 94-0899. REFERENCES 1. Nakajin S., Shimoda M., Haniu M., Shivery J. E. and Hall P. F.: C21 steroids side chain cleavage enzyme from porcine adrenal microsomes. J. Biol. Chem. 259 (1984) 3971-3976. 2. Zuber M. X., Simpson E. R. and Waterman M. R.: Expression of bovine 17ct-hydroxylase cytochrome P450 cDNA in nonsteroidogenic (Cos 1) cells. Science 234 (1986) 1258-1261. 3. Kagimoto M., Winter J. S. D., Kagimoto K., Simpson E. R. and Watweman M. R.: Structural characterization of normal and mutant human steroid 17ct-hydroxylasegenes: molecular bases of one example of combined 17ct-hydroxylase/17,20-1yase deficiency. Molec. Endocr. 2 (1988) 564-570. 4. Picado-Leonard J. and Miller W. L.: Cloning and sequence of the human gene for P450C17 (steroid 17~-hydroxylase/17,20lyase): similarity with the gene for P450c21. D N A 6 (1987) 439-448. 5. Sparkes R. S., Klisak I. and Miller W. L.: Regional mapping of genes encoding human steroidogenic enzymes: P450scc to 15q23-q24, adrenodoxin to 11q22; adrenodoxin reductase to 17q24-q25; and P450c17 to 10q24-q25. DN,4 10 (1991) 359-365. 6. Yanase T., Simpson E. R. and Waterman M. R.: 17ct-hydroxylase/17,20-1yase deficiency: from clinical investigation to molecular definition. Endocrine Rev. 12 (1991) 91-107. 7. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B. and Ehlirich H. A.: Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239 (1988) 487-491. 8. Yanase T., Kagimoto M., Matsui N., Simpson E. R. and Waterman M. R.: Combined 17ct-hydroxylase/17,20-1yasedeficiency due to a stop codon in the N-terminal region of 17ct-hydroxylase cytochrome P450. Molec. Cell. Endocr. 59 (1988) 249-253. 9. Kagimoto K., Waterman M. R., Kagimoto M., Ferresia P., Simpson E. R. and Winter J. S. D.: Identification of a common molecular basis for combined 17ct-hydroxylase/17,20-1yasedeficiency in two Mennonite families. Hum. Genet. 82 (1989) 285-286. 10. Imai T., Yanase T., Waterman M. R., Simpson E. R. and Pratt J. R.: Canadian Menonites and individuals residing in the Friesland region of the Netherlands share the same molecular basis of 17ct-hydroxylase deficiency. Hum. Genet. 82 (1989) 285-286. 11. Yanase T., Sanders D., Shibata A., Matsui N., Simpson E. R. and Waterman M. R.: Combined 17~-hydroxylase/17,20-1yase deficiency due to a 7 base pair duplication in the N-terminal region of the cytochrome P45017ct (CYP 17) gene. J. Clin. Endocr. Metab. 70 (1990) 1325-1329. 12. Biason A., Mantero F., Scaroni C., Simpson E. R. and Waterman M. R. Deletion within the CYP 17 gene together with insertion of foreign DNA is the cause of combined complete 17ct-hydroxylase/17,20-1yase deficiency in an Italian patient. Molec. Endocr. 5 (1991) 2037-2045. 13. Lin D., Harikrishna J. A., Moore C. C. D., Jones K. L. and Miller W. L.: Missense mutation serine~°~ proline causes 17ct-hydroxylase deficiency. J. Biol. Chem. 266 (1991) 15,992-15,999. 14. Rumsby G., Skinner C., Lee H. A. and Honour J. W.: Combined 17ct-hydroxylase/17,20-1yase deficiency caused by heterozygous stop codons in the cytochrome P450 17ct-hydroxylase gene. Clin. Endocr. 39 (1993) 483-485. 15. Fardella C. E., Zhang L-H., Mahachoklertwattana P., Lin D. and Miller W. L.: Deletion of amino acids Asp487-Ser4~sPhe4s9 in human cytochrome P450c17 causes severe 17ahydroxylase deficiency. J. Clin. Endocr. Metab. 77 (1993) 489-493. 16. Monno S., Ogawa H., Date T., Fujioka M., Miller W. L. and Kobayashi M.: Mutation of Histidine 373 to Leucine in

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17.

18.

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20.

21.

22.

cytochrome P450c 17 causes 17a-hydroxylase deficiency. J. Biol. Chem. 268 (1993) 25,811-25,817. Imai T., Globerman H., Gertner J. M., Kagawa N. and Waterman M. R.: Expression and purification of functional human 17a-hydroxylase/17,20-1yase (P450c17) in Escherichia coil J. Biol. Chem. 268 (1993) 19,681-19,689. Fardella C. E., Hum D. W., Homoki J. and Miller W. L.: Point mutation of Arg 440 to His in cytochrome P450cl 7 causes severe 17~-hydroxylase deficiency, ft. Clin. Endocr. Metab. 79 (1994) 160-164. Toyota N., Monno S. and Kobayashi T.: Two-bp deletion in exon 5 of CYP 17 gene in a family with 17~hydroxylase deficiency. Folia. Endocr. Japon. 69 (1993) 295 (Abstract). Yanase T., Kagimoto M., Suzuki S., Hashiba K., Simpson E. R. and Waterman M. R.: Deletion of a phenylalanine in the N-terminal region of human cytochrome P45017~ results in partial combined 17~-hydroxylase/17,20-1yase deficiency. J. Biol. Chem. 264 (1989) 18,076-18,082. Ahlgren R., Yanase T., Simpson E. R., Winter J. S. D. and Waterman M. R.: Compound heterozygous mutations (Arg 239 --* Stop, Pro342 ~ Thr) in the CYP17 (P45017~) gene lead to ambiguous external genitalia in a male patient with partial combined 17~-hydroxylase deficiency. J. Clin. Endocr. Metab. 74 (1992) 667-672. Yanase T., Waterman M. R., Zachmann M., Winter J. S. D., Simpson E. R. and Kagimoto M.: Molecular basis of apparent

23. 24. 25.

26. 27.

28.

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