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Department of Internal Medicine III (S.M.B.-P., P.N., W.W., H.V.), Division of Clinical Endocrinology and Metabolism, Medical University of Vienna, Section of Clinical Biometrics (G.H.), Medical Statistics and Informatics, Core Unit of the Medical University of Vienna, A-1090 Vienna, Austria
Address all correspondence and requests for reprints to: Sabina M. Baumgartner-Parzer, Ph.D., Department of Internal Medicine III, Division of Endocrinology, Metabolism, Waehringer Guertel 18-20, A-1090 Vienna, Austria. E-mail: sabina.baumgartner-parzer{at}meduniwien.ac.at.
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Abstract |
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Genotyping 400 unrelated alleles from 200 clinically unaffected individuals, this study revealed a carrier frequency of 9.5%, including so-called "classic" (5.5%) and "nonclassic" (4%) CYP21A2-gene aberrations. The observed heterozygosity for CAH in Yugoslavs was not different (P = 0.8095) from that in non-Yugoslavs.
In conclusion, the observed CAH carrier frequency of 9.5% suggests a higher prevalence of CAH heterozygosity in a middle European population than hitherto estimated independently of the individuals’ Yugoslav or non-Yugoslav origin.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The severity of the disease (phenotype) usually reflects the combination (compound heterozygosity) of more or less severe mutations and thus covers a wide range of disease phenotypes. Due to such overlap of disease phenotypes, an accurate differentiation between so-called classic (salt wasting and simply virilizing type) from nonclassic (late onset, less severe) forms of the disease (1, 2) is sometimes difficult. Although critical salt loss crises occur only in individuals with severe mutations, an activation of the renin-angiotensin system is also apparent in simple virilizers, although not in nonclassical cases (4).
So far, carrier frequency has been calculated applying the Hardy-Weinberg distribution (5) to data obtained from CAH newborn screening programs, which fail to reliably detect milder forms of the disease. On the basis of population genetic evaluation, hormonal data, and human leukocyte antigen genotyping, certain populations including Yugoslavs (1.6%) have been reported to exhibit a higher disease frequency (6, 7), particularly of the less severe form of CAH, than the general white population. In this context, it is of note that the Austrian population includes individuals of Germanic origin as well as migrants of Slavic background such as Czechs, Poles, and Yugoslavs, the latter representing at least 8.2% of the eastern Austrian population (http://www.magwien.gv.at/ma66/aktuell/bevoelkerung.htm).
So far, we are aware of only one study (8) that has determined CAH heterozygosity rates by CYP21A2 genotyping in an unselected population (randomly chosen New Zealand neonates). Although large deletions were not evaluated in this study (8), CAH heterozygosity frequency in New Zealanders was higher than expected based on genotype compared with that predicted from hormonal newborn screening.
Thus, the purpose of this investigation was to establish the frequency of heterozygous mutations of the CYP21A2 gene (carrier frequency) in a clinically asymptomatic population, not characterized by any metabolic abnormalities.
Therefore, the present study tested the hypothesis that: 1) carrier frequency for CAH in a middle European (Austrian) population might be higher than predicted from hormonal screening data; and 2) individuals of Yugoslav origin might exhibit a higher heterozygosity rate than a non-Yugoslav, middle European population.
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Subjects and Methods |
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We studied 400 independent alleles from 200 individuals registered at our Department of Internal Medicine III (University Hospital, Vienna, Austria). Each of these individuals was questioned and stated her/his origin from the former republic of Yugoslavia or that she/he had non-Yugoslav ancestors for the last two generations. Patients with signs of virilization, a family history of CAH, or adrenal diseases (e.g. adrenal tumors, Addison’s disease) were excluded from the study. Written informed consent for CYP21 mutation analysis was obtained from all individuals studied.
CYP21 mutation analysis
Genomic DNA was extracted from peripheral blood leukocytes (9, 10). Genotyping for large gene deletions and conversions was by Southern blot analysis as described previously (9, 10). In brief, TaqI and BglII digested DNAs, immobilized on nylon membranes, were hybridized with a 32P-deoxy CTP-labeled CYP21 probe (American Type Culture Collection). Heterozygous and homozygous deletions/conversions as well as duplications were distinguished on the basis of the intensities and ratios of TaqI and BglII fragments, as depicted and described previously in more detail (10, 11).
The common mutations P30L, I2Splice, I172N, Cluster E6, V281L, F307insT, G291S, Q318X, R356W, G424S, and P453S were detected by direct sequencing of three fragments (I-III), specifically amplified by selective PCR primers differentiating the functional CYP21A2 gene from the CYP21A1P pseudogene by the 8-bp deletion located in exon 3 of CYP21A1P, as depicted (10) and described previously in more detail (9, 10, 12). The GenBank accession no. is NM 000500.
Calculation of heterozygosity (13)
Assuming Hardy-Weinberg equilibrium, heterozygosity was calculated as H = A2 – a2, where A and a denote the relative frequencies of the two alleles. The assumption of Hardy-Weinberg equilibrium was tested by comparing expected and observed numbers of heterozygotes using a 
2 test.
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Results |
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The study population consisted of the Yugoslav and non-Yugoslav group, each including 100 unrelated individuals (80 females, 20 males) with a mean age of 44.8 ± 12.6 and 53.6 ± 15.9 yr, respectively. According to their diction, individuals of the Yugoslav group originated from Serbia (58%), Bosnia (18%), Croatia (15%), Voyvodina (6%), and Macedonia (3%).
CYP21 mutation analysis
CYP21 mutation analysis detected heterozygous CYP21A2 gene aberrations in 10 individuals (10%) of the Yugoslav and in nine subjects (9%) of the non-Yugoslav group (P = 0.8095, 2 test). Comparing observed and expected number of heterozygotes, no deviation from Hardy-Weinberg equilibrium could be detected (Yugoslavs: expected heterozygosity = 9.5%, P = 0.5987; non-Yugoslavs: expected heterozygosity = 8.6%, P = 0.6375).
In summary, this study revealed 9.5% (19 of 200 subjects) of individuals of a middle European (Austrian) population–independent of the subjects’ Yugoslav or non-Yugoslav origin (Table 1
)–to be carriers for CAH (due to 21-OH deficiency). The majority of CYP21A2 gene aberrations were single nucleotide substitutions (6.5%) compared with 3% apparent gene deletions/conversions (including the 8-bp deletion in exon 3). So-called nonclassic mutations were found in 4% of subjects in both groups, and classic mutations were present in 6% of Yugoslavs and in 5% of non-Yugoslavs.
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), being within the range reported previously (14). In that context, it is of note that deletion/duplication of the pseudogene (CYP21A1P) is of no clinical consequence. Thus, these CYP21A1P alterations were not included in the carrier count.
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Discussion |
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The determination of carrier frequency in a larger population is, at present, difficult, in regard to the currently available technique, which is both time-consuming and expensive. Although the sample size in our study was smaller, our data are in line with the study published by Fitness et al. (8), which, to our knowledge, is the only one performing CYP21A2 genotyping in randomly chosen New Zealand neonates blind to 17-OHP levels. In this unselected population, heterozygous CYP21A2 mutations were detected in 4.8% of individuals. The study by Fitness et al. (8) genotyped for the nine most common CYP21A2 mutations but was unable to identify heterozygous deletions in normal individuals. Thus, also in New Zealanders, the true heterozygosity rates will thus clearly be higher, because large deletions and certain types of conversions (detectable only by Southern blotting)–dependent on the population studied (9, 19, 20)–are assumed to represent up to 40% (20) of CAH alleles.
Certain ethnic populations including Yugoslavs are assumed to exhibit a higher frequency of such milder nonclassical forms of 21-hydroxylase deficiency (6, 7). The present study, however, shows a CAH carrier frequency of 10% for Yugoslavs and 9% for non-Yugoslavs, including so-called nonclassical mutations, which were detected in 4% of individuals in each group. Assuming that a higher disease frequency is reflected by a higher carrier frequency and vice versa, our data are in contrast to these previously published data (6, 7). This discrepancy may relate to the circumstance that in the original reports (6, 7), the frequency of nonclassic disease was estimated on the basis of pedigree analysis, human leukocyte antigen genotyping, and hormonal data, with CYP21 genotyping not yet available.
Nowadays, CYP21 genotyping reveals that CAH, in fact, is a more complex disease with combined heterozygosity for at about 70 different CYP21A2 gene defects, including single nucleotide substitutions, microconversions, and large deletion/conversion defects, which differently affect 21-OH enzymatic activity. Correlations between CYP21 genotype and phenotype have been studied in various populations (19, 21). CYP21 mutations were grouped into categories according to the level of enzymatic activity predicted from in vitro mutagenesis and expression studies (22). Characterizing the patients’ phenotype by 17-OHP levels or scores for signs of androgen excess, allelic variation of the CYP21 genotype (patients are usually compound heterozygotes for different mutations) accounts for at about 80% of phenotypic variation (23). Such discrepancy could also relate to so-called leakiness of splice mutations (leaving 1–2% of normally spliced mRNA sufficient to ameliorate enzymatic deficiency in some patients) and to genetic or environmental factors other than 21-hydroxylase activity, which may also influence the phenotype, as reviewed by Speiser and White (1, 11).
This complex background aggravates accurate definition of milder so-called nonclassic disease vs. classic disease phenotypes as well as vs. carriers of the disease and individuals not affected with CYP21A2 gene defects. In that context, it is of note that recent hormonal data from 21-OH newborn screening programs suggest CAH to be a continuum of disorders rather than one with discrete subtypes (18).
Recent evidence suggests that the currently used cut-off levels for ACTH-stimulated 17-OHP, established before molecular analysis was available, overestimate the diagnosis of nonclassic forms of the disease (24, 25). Thus, CYP21A2 genotyping is the only accurate method to define carriers for inherited CYP21 defects, as also recently shown in Slovenian hyperandrogenic women (26). In that context, it is of note that the subjects genotyped in the present study were asymptomatic with respect to CAH. Therefore, there was no indication for additional evaluation or treatment of these individuals.
However, all heterozygotes identified received careful genetic counseling in regard to the risk affecting their offspring.
In conclusion, the observed CAH carrier frequency of 9.5% suggests a higher prevalence of CAH heterozygosity in a middle European (Austrian) population than hitherto estimated, independent of the individuals’ Yugoslav or non-Yugoslav origin.
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Acknowledgments |
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Footnotes |
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Abbreviations: CAH, Congenital adrenal hyperplasia; 17-OHP, 17-hydroxyprogesterone.
Received August 30, 2004.
Accepted November 11, 2004.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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an V, Stopar-Obrea M, 
erjav-Tan
ek M, Breskvar K, Krinik C, Battelino T 2003 Mutational spectrum of congenital adrenal hyperplasia in Slovenian patients: a novel Ala15Thr mutation and Pro30Leu within a larger gene conversion associated with a severe form of the disease. Eur J Endocrinol 149:137–144[Abstract]
an V, Preelj J, Vidan-Jeras B, Breskvar K 1999 Adrenal 21-hydroxylase gene mutations in Slovenian hyperandrogenic women: evaluation of corticotrophin stimulation and HLA polymorphisms in screening for carrier status. Eur J Endocrinol 141:132–139[Abstract]This article has been cited by other articles:
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  R. Bergamaschi, C. Livieri, C. Uggetti, E. Candeloro, M. G. Egitto, A. Pichiecchio, V. Cosi, and S. Bastianello Brain white matter impairment in congenital adrenal hyperplasia. Arch Neurol, March 1, 2006; 63(3): 413 - 416. [Abstract] [Full Text] [PDF]
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