European Journal of Endocrinology (2000) 143 397±403 ISSN 0804-4643

CLINICAL STUDY Genotype±phenotype associations in non-classical steroid 21-hydroxylase de®ciency

Naomi Weintrob, Chaim Brautbar1, Athalia Pertzelan, Zeev Josefsberg, Zvi Dickerman, Arieh Kauschansky, Pearl Lilos, Dalia Peled, Moshe Phillip and Shoshana Israel1 Institute for Endocrinology and Diabetes, Schneider Children's Medical Center of Israel, Petah Tiqva and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel and 1Tissue Typing Unit, Hadassah Medical Organization, Jerusalem, Israel (Correspondence should be addressed to N Weintrob, Institute for Endocrinology and Diabetes, Schneider Children's Medical Center of Israel, 14 Kaplan Street, Petah Tiqva 49202, Israel; Fax: +972-3-9253836)

Abstract Objective: To evaluate whether genotype differences can explain the clinical variability of non-classical steroid 21-hydroxylase de®ciency (NC21-OHD) and to determine if genotype is related to ethnic origin. Design: Genotyping for mutations in the steroid 21-hydroxylase (CYP21) gene was performed in 45 unrelated Israeli Jewish patients (nine males) with NC21-OHD (60 min 17-hydroxyprogesterone (17- OHP), 45±386 nmol/l) who were referred for evaluation of postnatal or true precocious/ early . Eleven siblings diagnosed through family screening were genotyped as well. Methods: Patients were divided by genotype into three groups: (A) homozygous or compound heterozygous for the mild mutations (V281L or P30L) (n=29; eight males); (B) compound hetero- zygous for one mild and one severe mutation (Q318X, I2 splice, I172N) (n=12; no males); (C) mild mutation detected on one allele only (n=4; one male; peak 17-OHP 58±151 nmol/l). We then related the genotype to the ethnic origin, clinical phenotype and level. Since group C was very small, comparisons were made between groups A and B only. Results: At diagnosis, group B tended to be younger (5.863.0 vs 8.164.3 years, P ˆ 0.09), had greater height SDS adjusted for mid-parental height SDS (1.661.1 vs 0.761.4, P ˆ 0.034), tended to have more advanced bone age SDS (2.961.5 vs 1.762.1, P ˆ 0.10) and had a higher peak 17-OHP level in response to ACTH stimulation (226692 vs 126662 nmol/l, P < 0.01). Group B also had pubarche and gonadarche at an earlier age (5.162.4 vs 7.462.2 years, P < 0.01 and 7.461.8 vs 9.961.4 years, P < 0.001, respectively) and a higher rate of (50 vs 17%, P ˆ 0.04). Stepwise logistic regression analysis (excluding males) yielded age at gonadarche as the most signi®cant variable differentiating the two groups, with a positive predictive value of 86% for a cut-off of 7.5 years. Conclusions: The ®ndings suggest that genotype might explain some of the variability in the phenotypic expression of NC21-OHD. Compound heterozygotes for one mild and one severe mutation have a higher peak 17-OHP associated with and gonadarche at an earlier age and more frequent precocious puberty. Hence, the severity of the enzymatic defect might determine the timing and pattern of puberty.

European Journal of Endocrinology 143 397±403

Introduction and (C) non-classical (NC21-OHD), which is associated with different degrees of postnatal virilization develop- Congenital adrenal hyperplasia (CAH) denotes a family ing during childhood or at puberty and may be of disorders due to a defect in cortisol biosynthesis. asymptomatic (1). De®cient 21-hydroxylase (CYP21) activity, required The disorder is inherited as a monogenic autosomal to convert 17-hydroxyprogesterone (17-OHP) to 11- recessive trait. The CYP21 gene and a highly homo- deoxycortisol, is the most common cause of CAH and logous inactive pseudo-gene, CYP21P, are located on one of the most common genetic inborn errors of the short arm of chromosome 6 within the HLA class III metabolism. The disorder has traditionally been divided region. CYP21 gene deletion and at least ten sequence into three types according to severity of expression. (A) aberrations, probably transferred from CYP21P by gene Salt-wasting (SW) and (B) simple virilizing (SV); the conversion, have been identi®ed as causing the different more severe SW and SV forms are collectively referred to forms of 21-OHD (2). The functional consequences of as classical steroid 21-hydroxylase de®ciency (21-OHD), individual mutations have been studied, and the q 2000 Society of the European Journal of Endocrinology Online version via http://www.eje.org

Downloaded from Bioscientifica.com at 10/01/2021 08:31:10AM via free access 398 N Weintrob and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2000) 143 mutations have been classi®ed according to the degree 0.1 to 17.5 years (median, 7.25). Ethnic origin and of the resultant enzymatic inactivation, so that the clinical signs at presentation are shown in Table 1. For genotype can usually predict the clinical phenotype the second part of the study, the genotype and clinical (3, 4). Previous studies have correlated the genotype characteristics of 11 affected siblings (six males) with the three main forms of the disease, although data detected by family screening were compared with the on the correlation of the genotype with the various index case. The study protocol was approved by the clinical presentations of the non-classical form are Ethics Committee of Rabin Medical Center. We obtained lacking. informed consent from the patients and also from the The prevalence of NC21-OHD among Ashkenazi Jews parents of the children who were less than 18 years old. is remarkably high (3.2±3.7%) (5); about 80% carry Height of the patients and parents was measured with the mild mutation V281L (6). Screening for CYP21 the Harpenden±Holtain stadiometer. Auxological gene mutations in NC21-OHD in other Jewish ethnic results were expressed in terms of standard deviation groups has not been done. scores (SDS) for age, based on the standards of Tanner & In this study we screened for CYP21 gene mutations Whitehouse (8). Bone age (BA) was determined in Jewish patients with NC21-OHD to evaluate whether according to Greulich & Pyle (9). Height was accepted genotype differences might explain the clinical varia- as the ®nal height when the increment was < 1 cm over bility of this disorder and to determine whether there is 1 year or when BA was $15 years in girls and $17 an association between genotype and ethnic origin. years in boys. Clitoris and penile size was measured by sliding caliper. Clitoromegaly and macrogenitosomia were considered when measurements were > 2 S.D. for Subjects and methods age and Tanner stage (10, 11). We transformed the The genetic study was carried out between 1995 and measurements of clitoral size made from 1980 to 1992 1998 in patients who had been followed in our institute to the clitoral index published in 1992 (10). since the late 1970s. Puberty was assessed according to Marshall & Tanner The study population included 45 unrelated Jewish (12, 13). Pubarche was de®ned as Tanner 2 for pubic patients (nine males) with NC21-OHD who were hair (12, 13). The criterion of true puberty was referred for evaluation of postnatal virilization or true gonadarche: buds in girls and a testicular precocious/early puberty. The clinical diagnosis of volume reaching 4 ml in boys. True precocious puberty NC21-OHD was based upon presenting signs of post- was diagnosed when gonadarche appeared before the natal hyperandrogenism and the characteristic 60-min age of 8 years in girls and 9 years in boys. When response of 17-OHP to 0.25 mg adrenocorticotropin gonadarche was present, a -releasing (ACTH) stimulation (45±386 nmol/l) (7) with normal hormone (GnRH) stimulation test was performed levels of 11-deoxycortisol. Age at diagnosis ranged from concomitantly with the ACTH test. Plasma 17-OHP and cortisol levels were measured in the morning after an overnight fast before and 60 min after an i.v. bolus of a ®xed dose of 0.25 mg ACTH Table 1 Demographic and clinical characteristics of 45 Jewish patients with NC21-OHD. (7) (Synacthen, Ciba-Geigy, Basel, Switzerland). Levels of plasma luteinizing hormone (LH) and follicle- Patients stimulating hormone (FSH) were measured before and (n ˆ 45) 30 and 60 min after an i.v. bolus of 50 mg/m2 GnRH No. (%) (Relisorm; Serono, Aubonne, Switzerland). Pubertal LH $ Ethnicity* response was de®ned as 5 IU/l (14). Also determined Ashkenazi 24 (53) were basal levels of androstenedione, , 11- Sephardic 8 (18) deoxycortisol and . Measurements were per- Asian 1 (2) formed by radioimmunoassay (RIA) with commercial Combined 12 (27) kits (17-OHP, cortisol, testosterone, estradiol: DPC, Los Presenting sign² Precocious pubarche 20 (44) Angeles, CA, USA; androstenedione: Buhlman, Basel, True precocious puberty at diagnosis 12 (27) Switzerland; 11 deoxycortisol: Sigma, St Louis, MO, Clitoromegaly/macrogenitosomia 19 (42)³ USA; LH and FSH: chemiluminescent enzyme immu- PCO-like syndrome 3 (7) noassay; DPC). The plasma was separated and stored at Growth acceleration 3 (7) 8 Short ®nal height + S/P fast puberty 1 (2) 20 C until assayed. It is noteworthy that we have Hypertrichosis 1 (2) been using the DPC RIA for determination of 17-OHP and androgen levels since the mid-1980s. Before that, * By country of origin: Ashkenazi ± Eastern Europe and homemade kits were used and ®ndings were evaluated former USSR; Sephardic ± North Africa, Spain and the against norms established in our laboratory for each Balkans; Asian ± Yemen, Iraq and Iran. ² Most patients had more than one presenting sign. Tanner stage. Measurements done from the mid-1980s ³ 13/36 females (36%) and 6/9 males (66%). were compared with the norms evaluated by Lashansky PCO, polycystic ; S/P, status post. et al. (15). For each method, elevated androgen level www.eje.org

Downloaded from Bioscientifica.com at 10/01/2021 08:31:10AM via free access EUROPEAN JOURNAL OF ENDOCRINOLOGY (2000) 143 Phenotype±genotype associations in NC21-OHD 399 was de®ned as > 2 S.D. from the mean. Since the mutations (M) were as follows: androgen levels were determined by different methods, P30L, W: 50-CCACCTCCCGCCTCTTG-30 we did not compare the absolute levels between groups, P30L, M: 50-CCACCTCCTGCCTCTTG-30 but rather the frequency of the patients with elevated I2 splice, Wa: 50-AGCCCCCAACTCCTCCTGCA-30 androgen levels. I2 splice, Wc: 50-AGCCCCC ACCTCCTCCTGCA-30 Oral hydrocortisone (HC) therapy was instituted when I2 splice, M: 50-AGCCCCCAGCTCCTCCTGCA-30 there was evidence of progressive virilization, accelera- I172N, W: 50-CAGCATCATCTGTTACCTCA-30 tion of growth, true precocious puberty, advanced BA at I172N, M: 50-CAC ATCGTGGAGATGCAGCT-30 0 0 diagnosis ($1 S.D. for age) or an increase in DBA/Dage of cluster E6, W: 5 -CACAACGAGGAGAAGCAGCT-3 at least 6 months during follow-up. The dose (7.5± cluster E6, M: 50-CACAACGAGGAGAAGCAGCT-30 15 mg/m2, divided into two to three doses) was adjusted V281L, W: 50-AAGGGCACGTGCACATGGCT-30 to normalize growth and BA maturation rate (i.e. resume V281L, M: 50-AAGGGCACTTGCACATGGCT-30 the preacceleration growth curve) and to normalize Q318X, W: 50-CAGCGACTGCAGGAGGAGCT-30 morning plasma levels of testosterone and androstene- Q318X, M: 50-GCGACTGTAGGAGGAG-30 dione for age or Tanner stage. (Control DNAs for all the mutations analyzed were Eight girls were treated with i.m. GnRH agonist obtained as a gift from A Wedell.) In patients with (3.75 mg/4 weeks; Decapeptyl Depot; Ferring AB, homozygous deletion, the primers failed to anneal, and Malmo, Sweden) in addition to HC, for true precocious the speci®c PCR fragments were not obtained. A third puberty. The policy of the combined treatment evolved set of primers: CYP5: 50-GGTGCTGAACTCCAAGAGG-30 over several years, and therefore this regimen was and CYP16: 50-GTCCACAATTTGGATGGACCA-30 was introduced to only three girls of group B, four girls of used together with the sets above to obtain ampli®cation group A and one girl of group C; age at start of therapy in cases of homozygous deletion as a control for the was 6.7±8.5 years, age at which therapy was stopped reaction. was 9.5±12.5 years, and duration of therapy was 1.9± 3.5 years. Statistics Genetic analysis The data were analyzed with the BMPD program (18). Data are presented as means 6 S.D. unless otherwise The following mutations were sought in the patients, indicated. Between-group differences were analyzed by their parents and affected siblings: deletion of 8 bp in ANOVA or ANCOVA, as appropriate. The associations exon 3, ®ve point mutations: P30L exon 1, A/C655G between the clinical and laboratory characteristics and intron 2 (I2 splice), I172N exon 4, V281L exon 7, the genotype were analyzed with Pearson's chi-square Q318X exon 8 and a cluster of three point mutations in test or Fisher's exact test, as appropriate. Stepwise exon 6 (I236N+V237E+M239K). Blood samples were logistic regression was applied to estimate the best collected into EDTA and genomic DNA was isolated by predictor for differentiating between groups A and B the salting out method (16). Identi®cation of mutations (see below). was performed by the PCR sequence-speci®c oligonu- cleotide probe method. In brief, two sets of primers were used according to Wedell & Luthman (17) with some Results modi®cation. Set one ± CYP11: 50-GGAGCAATAAAG- The 90 alleles of the 45 unrelated Jewish subjects with GAGAAACTGA-30 and CYP48A: 50-GAGACTACTCCC- NC21-OHD contained the following disease-causing TGCTCTG-30. Set two ± CYP1: 50-TTCAGGCGATTC mutations: 72 (80%) V281L, six (6.7%) I2 splice, four AGGAAGGC-30 and CYP48: 50-CAGAGCAGGGAG- (4.4%) Q318X, two (2.2%) P30L, and two (2.2%) TAGTCTC-30. I172N; in four alleles (4.4%), none of the above The two PCR products were blotted onto nylon mutations was detected. Figure 1 shows the genotype membranes and hybridized with sequence speci®c distribution among the study patients. oligonucleotide probes which were end-labeled with Previous studies relating mutations to level of digoxigenin ddUTP (Boehringer Mannheim Biochem- enzymatic activity (19, 20) have shown that the mild icals, Mannheim, Germany). The membranes were then mutations (V281L and P30L) result in an enzyme with washed with 3 mol/l tetramethylammonium chloride 20±50% of normal activity, and the severe mutations solution at increasing temperatures ranging between (I2 splice, Q318X and I172N) in an enzyme with 0±2% 48 8C and 57 8C. Alkaline phosphatase-bound anti- of normal activity. Based on these ®ndings, we divided digoxigenin was used with the chemiluminescent the patients into three groups: (A) homozygous or substrate for alkaline phosphate (CDP star; Boehringer compound heterozygous for the mild mutations (n=29; Mannheim) for chemiluminescence detection. The eight males); (B) compound heterozygous for one mild oligonucleotide probes used for detection for wild-type and one severe mutation (n=12; no males); and (C) mild sequence (W) (including two wild-type polymorphic mutation detected on one allele only (n=4; one male; forms, Wa and Wc, for the I2 splice mutations) and peak 17-OHP 58±151 nmol/l).

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Figure 1 Genotype distribution among 45 patients with NC21-OHD.

Table 2 Clinical and laboratory characteristics of the study population by genotype (means6S.D.).

Signi®cance of Group A Group B Group C difference Parameter (n ˆ 29) (n ˆ 12) (n ˆ 4) (P a)

At diagnosis Age (years) 8.1 6 4.3 5.8 6 3.0 5.9 6 3.4 0.09 Bone age SDS 1.7 6 2.1 2.9 6 1.5 0.7 6 0.7 0.10 Height SDS 0.7 6 1.4 1.6 6 1.1 1.3 6 0.6 0.034b Basal 17-OHP (nmol/l) 40 6 39 46 6 31 9 6 6NS Peak* 17-OHP (nmol/l) 126 6 62 226 6 92 105 6 42 < 0.01 Therapy Starting age (years) 8.7 6 3.8 6.4 6 2.8 8.9 6 3.6 0.07 HC dose (mg/m2 per day) 14.9 6 4.2 14.9 6 3.8 9.7 6 3.5 NS Puberty Age at pubarche (years) Whole group 7.4 6 2.2 5.1 6 2.4 7.7 6 2.4 < 0.01 Females only 7.2 6 2.9 5.1 6 2.4 0.03 Age at gonadarche (years) Whole group 9.9 6 1.4 7.4 6 1.8 8.0 6 2.7 < 0.001 Females only 9.7 6 1.5 7.4 6 1.8 0.002 Menarchec 12.5 6 1.4 12.2 6 1.0 11.2 NSc Final height SDSd 0.53 6 0.9 1.3 6 0.9 0.5 6 0.2 NSb Mid-parental height SDS 0.2 6 0.6 0.3 6 1.3 0.1 6 0.2 NS

Group A ˆ homozygous or compound heterozygous for the mild mutations, group B ˆ compound heterozygous for one mild and one severe mutation and group C ˆ mild mutation (V281L) detected on one allele only. a Between A and B only. b Adjusted for mid-parental height. c No. of girls: 17, 7 and 1 for groups A, B and C respectively. d No. of subjects: 22, 5 and 2 for groups A, B and C respectively. * 60 min after ACTH stimulation. NS, not signi®cant. www.eje.org

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Table 3 Frequency of various parameters by genotype.

Group A Group B Group C Signi®cance of (n ˆ 29) (n ˆ 12) (n ˆ 4) association Parameter No. (%) No. (%) No. (%) (P a)

Gender Male 8 (28) 0 (0) 1 (25) 0.08 Female 21 (72) 12 (100) 3 (75) Ethnicity Ashkenazi 17 (59) 6 (50) 1 (25) NS Sephardic and Oriental 3 (10) 4 (33) 2 (50) Mixed 9 (31) 2 (17) 1 (25) Clitoromegaly/macrogenitosomia 12 (41) 6 (50) 1 (25) NS Precocious puberty at diagnosis* 5 (17) 6 (50) 2 (50) 0.04 Elevated androgens² 21 (72) 11 (92) 3 (75) NS HC therapy 26 (89) 11 (92) 3 (75) NS

Group A ˆ homozygous or compound heterozygous for the mild mutations, group B ˆ compound heterozygous for one mild and one severe mutation and group C ˆ mild mutation (V281L) detected on one allele only. a Between A and B only. * Group A, four females and one male; group B, females only. ² Androstenedione and/or testosterone > 2 S.D. for age and pubertal stage.

The characteristics of the study population by Ethnicity genotype group are shown in Tables 2 and 3. The Among the whole cohort, 44 patients (98%), including correlation of the 17-OHP level to group yielded similar all patients of Ashkenazi origin, carried at least one basal levels in groups A and B and a signi®cantly lower allele with the V281L mutation (Fig. 1). Two patients, level in group C (P < 0.05, Bonferroni correction for one of Yemenite origin and the other of mixed multiple comparisons) (Table 2); however, mean peak Yemenite±Ashkenazi origin, carried the P30L mutation 17-OHP was similar in groups A and C, and signi®- (21). In both cases, the mutation was carried by parents cantly higher in group B (P < 0.01 for B vs A and B vs C). of Yemenite origin. Because group C was very small, it was excluded There was no signi®cant difference in the ethnic from the statistical analysis of the other clinical group distribution between group A and group B characteristics. We found that at diagnosis, compared (Table 3). In group A patients the non-Ashkenazi with group A, group B tended to be younger comprised only 10% compared with 59% of Ashkenazi (P ˆ 0.09), had a greater height SDS adjusted for but the difference did not reach statistical signi®cance. mid-parental height SDS (P ˆ 0.034) and tended to ˆ have a more mature BA SDS (P 0.10). The number of Family screening patients with clitoromegaly or macrogenitosomia detected at diagnosis was similar in the two groups, Eleven siblings (six males, ®ve females) of ten families as was the number of patients with elevated androgen were diagnosed as having NC21-OHD according to levels, the number of treated patients (Table 3) and the 17-OHP response to ACTH stimulation (basal 17-OHP dose of HC/m2 (Table 2). > 8.5 nmol/l, peak 17-OHP > 58 nmol/l). All were Group B had pubarche and gonadarche (Table 2) at diagnosed at an earlier age than their index case an earlier age than group A (P < 0.01 and P < 0.001 owing to the family screening. Eight displayed similar respectively) and a higher rate of precocious puberty degrees of disease manifestations and three are still (P ˆ 0.04) (Table 3). Since there were no males in group asymptomatic at the age of 6.5 (V281L/I2 splice), 7.0 B, comparisons of age at pubarche and gonadarche and 9.0 (V281L/V281L) years; we do not yet know if were also done separately for girls, and yielded similar they have the cryptic form or will develop symptoms in results (Table 2). the coming years. All siblings shared the same genotype. Final height SDS, adjusted for mid-parental height Six siblings (two of the same family) were homozygous for SDS (Table 2), was similar between the two groups, but V281L, three were compound heterozygous for V281L/I2 only 22 patients of group A and ®ve of group B had splice, and two were heterozygous for V281L. reached ®nal height by the time of the study. A search for the mutations of the index cases among Application of stepwise logistic regression analysis their parents yielded 83 carriers of one mutation (in (excluding males) using the variables that showed four parents, no mutation was detected) and three signi®cance revealed that age at gonadarche was the parents (two fathers and one mother) of group 1 most signi®cant variable differentiating between the two patients who were homozygous for V281L; all were groups, with a positive predictive value of 86% for a cut- Ashkenazi. One father had a history of short stature, off of 7.5 years. and his height was 162 cm. His treated daughter and

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Downloaded from Bioscientifica.com at 10/01/2021 08:31:10AM via free access 402 N Weintrob and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2000) 143 son with NC21-OHD are 162 and 175 cm respectively, Both resumed regular menses on treatment with low-dose and both are homozygous for V281L. The other father is dexamethasone, and one who sought con- 167 cm tall and ®rst shaved at 15 years of age. The ceived on dexamethasone without any additional therapy. mother is 160 cm tall and hirsute. She had at Of note is the high frequency (50%) of true central age 13 and three spontaneous . precocious puberty (CPP) among patients with com- pound heterozygosity for one mild and one severe mutation (group B). True CPP has been described Discussion previously in classical 21-OHD (29), NC21-OHD (22), In agreement with previous studies, the majority of our and virilizing adrenal tumors (30), either at presenta- Jewish NC21-OHD patients were of Ashkenazi origin (5, tion or following institution of therapy when the 22) and almost all carried at least one allele with the hyperandrogenism resolved. The presentation of CPP V281L mutation (4, 6, 23). The P30L mutation (21) associated with hyperandrogenism usually correlates was found only in two patients, both of Yemenite origin, with advanced BA to the range of the pubertal years probably secondary to founder effect. The distinct (29, 30). Our group B patients did tend to have more genetic background of the Yemenite Jews has been advanced BA SDS at diagnosis than group A (Table 2), shown in studies of many other inherited disease (24). but only three out of six had a BA of > 8 years at Our results suggest that some of the variability in the gonadarche, which appeared before initiation of ther- clinical spectrum of NC21-OHD can be explained by apy, i.e. before androgen suppression. Therefore, it is genotype differences. The patients with compound probably the mildly elevated androgen levels that heterozygosity for one mild (either V281L or P30L) imitate physiologic and prematurely prime and one severe (Q318X, I2 splice, I172N) mutation the hypothalamic±pituitary±gonadal axis. Since CPP is (group B) had a more severe clinical picture than those quite frequently a presenting sign of NC21-OHD (27% in who carried two mild mutations (group A). At our group), we suggest that the ACTH stimulation test presentation, they were younger (all were diagnosed should be added to its evaluation. in the ®rst decade) and had a greater height SDS In four patients with peak 17-OHP levels within the adjusted for mid-parental height SDS and slightly more accepted range for NC21-OHD (105642 nmol/l), a mild mature BA. They also had earlier age at pubarche and mutation (V281L) was detected on one allele only gonadarche, a higher incidence of true central pre- (group C). Their mean basal 17-OHP level was, indeed, cocious puberty, and a higher peak 17-OHP in response signi®cantly lower than that of groups A and B (Table to ACTH stimulation. Thus, even though compound 2), but their mean peak 17-OHP level was compatible heterozygotes typically exhibit phenotypes correspond- with NC21-OHD (7) and was similar to that of group A. ing to the less severely affected allele (20, 25), their We did not perform screening for more mutations clinical course seems to be more severe than that of (R356W, P453S), or direct sequencing to detect rare patients carrying two mild mutations. Furthermore, alleles, owing to technical limitations. In other large while all the patients of group B presented in the ®rst studies screening for mutations in patients with clinical decade with either premature/early pubarche, preco- and laboratory evidence of 21-OHD (3, 4), mutations cious/early puberty, clitoromegaly or growth accelera- were not observed in 5±10% of chromosomes in which tion, the presentation of group A subjects was much they were expected according to the clinical status of the more variable; all the postpubertal patients belonged to index case. However, sequencing of the CYP21 gene was this group, as well as the three `asymptomatic' parents not done and, therefore, rare mutations might have who were detected by family screening. The explana- been missed. Since genotyping for all the mutations tions for the phenotypic heterogeneity among indivi- described so far, or direct sequencing, is expensive, duals carrying the same mutations are still hypothetical laborious and restricted to only a few research centers, and include the presence of other still unidenti®ed the diagnosis of 21-OHD de®ciency should probably still mutations, the activity of other genes encoding proteins rely on peak 17-OHP level in response to ACTH with extra-adrenal 21-hydroxylase activity (26), indivi- stimulation (7, 25). dual variation in the amount of protein produced (27), We believe that genotyping is justi®ed in patients with differences in intra-adrenal concentrations of progester- NC21-OHD owing to the existence of a subgroup with one (2), and differences in sensitivity to androgens (28). compound heterozygosity for one mild and one severe Our results are in agreement with Rumsby et al. (23) mutation. These ®ndings have important implications who studied mainly adult females with NC21-OHD and for future genetic and prenatal counseling, since found more frequent oligomenorrhea/amenorrhea and prenatal treatment with dexamethasone for fetuses infertility in those with one mild and one severe carrying two severe mutations is now feasible (31). mutation, and mostly regular menses in those homo- zygous for the mild mutation. Among our patients were two females, both homozygous for V281L, who Acknowledgements presented during adolescence with severe polycystic We are grateful to Dr Anna Wedell of Karolinska ovary syndrome with hirsutism and oligomenorrhea. Hospital, Stockholm, Sweden for providing the initial www.eje.org

Downloaded from Bioscientifica.com at 10/01/2021 08:31:10AM via free access EUROPEAN JOURNAL OF ENDOCRINOLOGY (2000) 143 Phenotype±genotype associations in NC21-OHD 403 help with the primers; and to Mrs Charlotte Sachs and 16 Miller SA, Dykes DD & Polesky HF. A simple salting out procedure Mrs Gloria Ginzach of the Editorial Board, Rabin for extracting DNA from human nucleated cells. Nucleic Acids Research 1988 16 1215. Medical Center, Beilinson Campus, and Oren Moran, 17 Wedell A & Luthman H. Steroid 21-hydroxylase (P450c21): a for their assistance. new allele and spread of mutations through the pseudogene. Human Genetics 1993 91 236±240. 18 BMPD Statistical Software. Ed. WJ Dixon. Los Angeles: University References of California Press, 1990. 19 Tusie-Luna MT, Traktman P & White PC. 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