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An Overview of Molecular Diagnosis of Steroid 21-Hydroxylase Deficiency

2001; Elsevier BV; Volume: 3; Issue: 2 Linguagem: Inglês

10.1016/s1525-1578(10)60651-4

1943-7811

Catherine E. Keegan, Anthony A. Killeen,

Metabolism and Genetic Disorders

Steroid 21-hydroxylase deficiency is the most frequent cause of congenital adrenal hyperplasia (CAH), a group of inborn errors of adrenal steroid biosynthesis.1White PC Speiser PW Congenital adrenal hyperplasia due to 21-hydroxylase deficiency.Endocr Rev. 2000; 21: 245-291Crossref PubMed Scopus (1121) Google Scholar Approximately 1 in 14,000 newborns is affected with the classic form of the disease. A milder, late-onset or non-classic form that is clinically evident in approximately 1 in 1000 females also exists. Both forms are the result of mutations at the same locus and are inherited as autosomal recessive traits. Very rarely, 21-hydroxylase deficiency can result from uniparental disomy.2Lopez-Gutierrez AU Riba L Ordonez-Sanchez ML Ramirez-Jimenez S Cerrillo-Hinojosa M Tusie-Luna MT Uniparental disomy for chromosome 6 results in steroid 21-hydroxylase deficiency: evidence of different genetic mechanisms involved in the production of the disease.J Med Genet. 1998; 35: 1014-1019Crossref PubMed Scopus (28) Google Scholar The primary laboratory means of diagnosis of this disease is by measurement of serum 17-hydroxyprogesterone, which is elevated in subjects with deficiency of this enzyme. This testing is also used in newborn screening programs that screen for 21-hydroxylase deficiency. Although biochemical testing remains the mainstay of diagnosis, molecular methods have acquired an essential role for identification of mutations and for assessment of at-risk pregnancies. This article provides an overview of published analytical methods for molecular diagnosis of steroid 21-hydroxylase deficiency. Approximately 90% of CAH result from deficiency of adrenal steroid 21-hydroxylase. Owing to a deficiency of this enzyme activity, there is a reduction in the ability of the adrenal cortices to produce physiological quantities of adrenal glucocorticoids, primarily cortisol. Synthesis of mineralocorticoids is also reduced, but because of the relatively smaller physiological requirement for mineralocorticoids, primarily aldosterone, impairment of enzyme function that does not abolish all activity may not be manifest as overt salt-wasting. Salt-wasting results from mineralocorticoid insufficiency at the distal renal tubule where sodium is reabsorbed from the glomerular filtrate and potassium and hydrogen ions are excreted. This syndrome results in loss of body sodium and water, metabolic acidosis, and the hyponatremia and hyperkalemia characteristic of adrenal insufficiency. Clinically, the disorder is characterized by life-threatening dehydration, vomiting, and diarrhea in the first few weeks of life. Approximately one-third of patients with classic CAH are able to produce enough mineralocorticoid to prevent salt-wasting. The other major biochemical feature of CAH is increased production of adrenal androgens. These compounds, which do not require 21-hydroxylase activity for their synthesis, provide the outlet for steroid synthesis in the adrenal gland. Owing to the lack of negative feedback that cortisol normally provides to the pituitary and hypothalamus, ACTH levels are elevated, which, in turn, stimulates the adrenal cortices. This stimulation causes hyperplasia of the glands and, combined with the metabolic block, results in high levels of production of adrenal androgens. This effect occurs in utero and results in variable degrees of virilization of the external genitalia of affected female fetuses and newborns. Affected males generally appear normal at birth. The mainstay of therapy is replacement of the missing steroids, electrolytes, and fluids. Surgical correction of the external genital abnormalities is commonly required in affected females. The functional CYP21 gene and the non-functional pseudogene, CYP21P, are located on the short arm of chromosome 6 in the class III region of the major histocompatibility complex (MHC).3Higashi Y Yoshioka H Yamane M Gotoh O Fujii-Kuriyama Y Complete nucleotide sequence of two steroid 21-hydroxylase genes tandemly arranged in human chromosome: a pseudogene and a genuine gene.Proc Natl Acad Sci USA. 1986; 83: 2841-2845Crossref PubMed Scopus (502) Google Scholar, 4White PC New MI Dupont B Structure of human steroid 21-hydroxylase genes.Proc Natl Acad Sci USA. 1986; 83: 5111-5115Crossref PubMed Scopus (503) Google Scholar The functional gene is a member of the cytochrome P450 family that specifically hydroxylates C21 on steroid precursors in the adrenal cortex. Both genes are normally in tandem arrangement with the genes encoding complement proteins 4A and 4B (Figure 1). It is generally believed that this pattern results from an ancient gene duplication event. Similar arrangements of 21-hydroxylase and C4 genes are found in several other mammalian species, suggesting that the duplication event preceded the radiation of mammals in evolution and implying that it occurred at least 60 million years ago. The CYP21P pseudogene usually contains a number of deleterious mutations that render it non-functional. These include a premature splice mutation in intron 2, a 8-bp deletion in exon 3, a series of point mutations that cause several missense mutations in exon 6, and a non-sense mutation in exon 8. Mutations that are commonly found in CYP21 are shown in Table 1. The majority of mutations that appear in CYP21 are either gene conversions of deleterious sequences normally found in CYP21P, or they are deletions. The severity of the phenotype generally depends on the mutations present on both alleles, but the phenotype is not entirely consistent among unrelated patients with the same mutations, or even among siblings of the same genotype. In general, gene deletions, the 8-bp deletion in exon 3, the exon 6 cluster of mutations, and Arg356Trp are associated with salt-wasting. The intron 2 mutation can be found in either salt-wasting or simple virilizing disease. Ile172Asn is associated with simple virilization, whereas Pro30Leu and Val281Leu are commonly seen in patients with non-classic disease. Based on consideration of the expected enzyme activity of the less severely impaired allele, it is possible to predict the phenotype with reasonable, but not complete, certainty.5Wilson RC Mercado AB Cheng KC New MI Steroid 21-hydroxylase deficiency: genotype may not predict phenotype.J Clin Endocrinol Metab. 1995; 80: 2322-2329Crossref PubMed Google ScholarTable 1Frequent Mutations in CYP21 in Classic CAHMutationLocationApproximate frequency (%)DeletionCYP2125–30Pro30LeuExon 15–10A(C)656GIntron 220–25Δ8bpExon 35–10Ile172AsnExon 45–10Exon 6 cluster*Exon 6 cluster includes Ile236Asn, Val237Glu, and Met239Lys. Data are based on frequencies reported in Reference 36.Exon 65–10Val281LeuExon 75–101757+TExon 7<5Gln318StopExon 85–10Arg356TrpExon 810* Exon 6 cluster includes Ile236Asn, Val237Glu, and Met239Lys. Data are based on frequencies reported in Reference 36. Open table in a new tab The initial report of linkage of human leukocyte antigen (HLA) serotypes with 21-hydroxylase deficiency indicated the approximate position of the locus.6Dupont B Oberfield S Smithwick E Lee T Levine L Close genetic linkage between HLA and congenital adrenal hyperplasia (21-hydroxylase deficiency).Lancet. 1977; 2: 1309-1311Abstract PubMed Scopus (287) Google Scholar Linkage-based diagnostic methods have been reported using various markers in the MHC. HLA serotyping7Pollack MS Maurer D Levine LS New MI Pang S Duchon M Owens RP Merkatz IR Nitowsky BM Sachs G Dupont B Prenatal diagnosis of congenital adrenal hyperplasia (21-hydroxylase deficiency) by HLA typing.Lancet. 1979; 1: 1107-1108Abstract PubMed Scopus (96) Google Scholar provided a mechanism to track mutant alleles within families but could not be easily applied to prenatal samples obtained by amniocentesis. However, serotyping revealed the existence of important linkage disequilibrium between certain HLA haplotypes and 21-hydroxylase deficiency. One of these that involves the HLA haplotype A3, B47, DR7, is associated with a 30-kb deletion that has removed most of the CYP21 functional gene and the adjacent C4Bgene, and left a non-functional hybrid gene composed of 5′ sequence from CYP21P and 3′ sequence from CYP21.8White PC Vitek A Dupont B New MI Characterization of frequent deletions causing steroid 21-hydroxylase deficiency.Proc Natl Acad Sci USA. 1988; 85: 4436-4440Crossref PubMed Scopus (210) Google Scholar Molecular approaches to performing HLA genotyping signaled the first DNA methods suitable for linkage-based prenatal diagnosis. These methods involved the use of various class I DNA probes, a HLA-B specific probe, and HLA class II probes.9Mornet E Boue J Raux-Demay M Couillin P Oury JF Demuz Y Dausset J Cohen D Boue A First trimester diagnosis of 21-hydroxylase deficiency by linkage analysis to HLA-DNA probes and by 17-hydroxyprogesterone determination.Hum Genet. 1986; 73: 358-364Crossref PubMed Scopus (57) Google Scholar, 10Strachan T Sinnott PJ Smeaton I Dyer PA Harris R Prenatal diagnosis of congenital adrenal hyperplasia.Lancet. 1987; 2: 1272-1273Abstract PubMed Scopus (24) Google Scholar, 11Killeen AA Seelig S Ulstrom RA Orr HT Diagnosis of classical steroid 21-hydroxylase deficiency using an HLA-B locus-specific DNA-probe.Am J Med Genet. 1988; 29: 703-712Crossref PubMed Scopus (15) Google Scholar, 12Pang S Pollack MS Loo M Green O Nussbaum R Clayton G Dupont B New MI Pitfalls of prenatal diagnosis of 21-hydroxylase deficiency congenital adrenal hyperplasia.J Clin Endocrinol Metab. 1985; 61: 89-97Crossref PubMed Scopus (60) Google Scholar These probes were used to test restriction fragment length polymorphisms (RFLPs) on Southern blots and took at least several days to perform. As with any linkage-based diagnostic strategy, errors can arise from recombination events between the marker locus and the disease locus, non-paternity, new mutation or back mutation, or mistaken clinical diagnosis in the proband. In addition, linkage-based approaches are only applicable to families in which an informative polymorphism can be identified. More recently, diagnostic approaches based on linkage have used polymorphic microsatellite markers in the region that can be typed by polymerase chain reaction (PCR).13Ramsden SC Sinnott PJ Characterization of gene rearrangements and gene conversion events in the 21-hydroxylase gene.in: Elles R Molecular Diagnostics of Genetic Diseases. Humana Press, Totowa, NJ1996: 121-140Crossref Google Scholar, 14Lako M Ramsden S Campbell RD Strachan T Mutation screeening in British 21-hydroxylase deficiency families and development of novel microsatellite based approaches to prenatal diagnosis.J Med Genet. 1999; 36: 119-124PubMed Google Scholar, 15Fitness J Dixit N Webster D Torresani T Pergolizzi R Speiser PW Day DJ Genotyping of CYP21, linked chromosome 6p markers, and a sex-specific gene in neonatal screening for congenital adrenal hyperplasia.J Clin Endocrinol Metab. 1999; 84: 960-966Crossref PubMed Scopus (101) Google Scholar The principal advantage of testing these is the ability to type the marker more rapidly than is possible by using Southern blot analysis for RFLPs. Our laboratory has identified several intragenic single nucleotide polymorphisms (SNPs) in intron 2 and in intron 6 of CYP21and CYP21P.16Killeen AA Jiddou RR Sane KS Characterization of frequent polymorphisms in intron 2 of CYP21: application to analysis of segregation of CYP21 alleles.Clin Chem. 1998; 44: 2410-2415PubMed Google Scholar, 17Jiddou RR Wei WL Sane KS Killeen AA Single-nucleotide polymorphisms in intron 2 of CYP21P: evidence for a higher rate of mutation at CpG dinucleotides in the functional steroid 21-hydroxylase gene and application to segregation analysis in congenital adrenal hyperplasia.Clin Chem. 1999; 45: 625-629PubMed Google Scholar These can provide information on the inheritance of alleles in families and are also useful in identifying deletions that appear as apparent non-transmission of an allele by a parent. The most widely used method to detect the relatively common deletion of CYP21 in affected families is Southern blot analysis. The use of restriction enzymes that generate separate fragments that contain CYP21 and CYP21P allows for estimation of the relative dosage of CYP21. Standard restriction enzymes used to digest genomic DNA include TaqI and BglII. The expected sizes of restriction fragments generated from these digestions are shown in Table 2. It is also useful to probe Southern blots for the neighboring C4 genes because deletions of CYP21 frequently include C4B. TaqI-restricted DNA can be simultaneously probed for both the C4 genes and the 21-hydroxylase genes because of the non-overlapping RFLPs generated. DNA probes for Southern blot analysis for the 21-hydroxylase and C4 genes are available from the American Type Culture Collection (Manassas, VA).Table 2Expected RFLP Sizes of CYP21 and CYP21P after Restriction DigestionEnzymeCYP21CYP21PTaqI3.7 kb3.2 kbBglII10 kb12 kb Open table in a new tab In TaqI-restricted genomic DNA, CYP21 and CYP21P are located on 3.7-kb and 3.2-kb restriction fragments, respectively. Subjects with heterozygous deletions of CYP21 can be identified by decreased intensity of the 3.7-kb fragment relative to that of the 3.2-kb fragment. Subjects with homozygous CYP21 deletion show absence of the 3.7-kb fragment. Difficulties with interpretation of Southern blots can arise from some of the duplication and deletion patterns that can be found at the 21-hydroxylase loci. Deletions of CYP21P are fairly common, being present in 9 to 14% of Caucasian chromosomes.17Jiddou RR Wei WL Sane KS Killeen AA Single-nucleotide polymorphisms in intron 2 of CYP21P: evidence for a higher rate of mutation at CpG dinucleotides in the functional steroid 21-hydroxylase gene and application to segregation analysis in congenital adrenal hyperplasia.Clin Chem. 1999; 45: 625-629PubMed Google Scholar, 18Morel Y Andre J Uring-Lambert B Hauptmann G Betuel H Tossi M Forest MG David M Bertrand J Miller WL Rearrangements and point mutations of P450c21 genes are distinguished by five restriction endonuclease haplotypes identified by a new probing strategy in 57 families with congenital adrenal hyperplasia.J Clin Invest. 1989; 83: 527-536Crossref PubMed Scopus (109) Google Scholar Because the dosage of CYP21 is determined by comparing its signal strength to that of CYP21P on Southern blots, it follows that a subject with deletions of both CYP21 and CYP21P could be mistakenly thought to have a normal gene dosage.13Ramsden SC Sinnott PJ Characterization of gene rearrangements and gene conversion events in the 21-hydroxylase gene.in: Elles R Molecular Diagnostics of Genetic Diseases. Humana Press, Totowa, NJ1996: 121-140Crossref Google Scholar Subjects with a duplication of either 21-hydroxylase gene might also give unexpected results on gene dosage studies by Southern blot analysis. These issues emphasize the need for careful family studies when evaluating a subject for gene deletions.1White PC Speiser PW Congenital adrenal hyperplasia due to 21-hydroxylase deficiency.Endocr Rev. 2000; 21: 245-291Crossref PubMed Scopus (1121) Google Scholar Pulsed-field gel electrophoresis may provide additional information on gene dosage in unusual cases.19Collier S Sinnott PJ Dyer PA Price DA Harris R Strachan T Pulsed field gel electrophoresis identifies a high degree of variability in the number of tandem 21-hydroxylase and complement C4 repeats in 21-hydroxylase deficiency haplotypes.EMBO J. 1989; 8: 1393-1402PubMed Google Scholar As with many genetic diseases, PCR has been widely adopted for amplification of the locus before specific mutation identification. Owing to the presence of the highly homologous pseudogene, amplification of CYP21 is not straightforward. Primers must be selected that recognize sequences in CYP21 that are not present in CYP21P. These primer binding sites must be sufficiently specific for CYP21 such that CYP21Pcannot be amplified in the PCR through promiscuous priming. These requirements severely limit the number of CYP21-specific primer targets. Most authors have exploited two regions in which CYP21 differs from CYP21P. These are the 8-bp sequence in exon 3 that is present in CYP21 but deleted in CYP21P, and the exon 6 cluster of mutations that is composed of four nucleotide differences between the functional gene and pseudogene. Using this approach, CYP21 can be amplified in two overlapping fragments as shown in Figure 2. After amplification, a variety of methods to detect the mutations present can be used. Several caveats involving PCR amplification of CYP21 need to be considered. First, several studies have reported the inability to amplify both CYP21 alleles, particularly around intron 2, the site of the important premature splice mutation, A655G or C655G. This can result in subjects who are heterozygous for this mutation appearing to be homozygous.20Day DJ Speiser PW Schulze E Bettendorf M Fitness J Barany F White PC Identification of non-amplifying CYP21 genes when using PCR-based diagnosis of 21-hydroxylase deficiency in congenital adrenal hyperplasia (CAH) affected pedigrees.Hum Mol Genet. 1996; 5: 2039-2048Crossref PubMed Scopus (120) Google Scholar A report indicates that this artifact may be overcome through the use of a blend of DNA polymerases that includes both Taq polymerase and Pwopolymerase.21Schulze E Bettendor M Maser-Gluth C Decker M Schwabe U Allele-dropout using PCR-based diagnosis for the splicing mutation in intron-2 of the CYP21B-gene: successful amplification with a TAQ/PWO-polymerase mixture.Endocr Res. 1998; 24: 637-641Crossref PubMed Scopus (20) Google Scholar Careful examination of the mutational status of family members and of the segregation of linked microsatellites may be useful in identifying this artifact. Second, it is clear that CYP21 sequences can be back-converted to CYP21P.22Day DJ Speiser PW White PC Barany F Detection of steroid 21-hydroxylase alleles using gene-specific PCR and a multiplexed ligation detection reaction.Genomics. 1995; 29: 152-162Crossref PubMed Scopus (109) Google Scholar This brings sequences from CYP21 into the pseudogene. If CYP21 primer binding sequences were introduced into CYP21P, such conversion could conceivably lead to unintended PCR priming of CYP21P and mistaken identification of mutations that could be assumed to represent gene conversion events in CYP21. Additional useful information may be obtained by analysis of overlapping PCR products and by probing Southern blots with gene-specific oligonucleotide probes for the primer-binding regions.23Killeen AA Sane KS Orr HT Molecular and endocrine characterization of a mutation involving a recombination between the steroid 21-hydroxylase functional gene and pseudogene.J Steroid Biochem Mol Biol. 1991; 38: 677-686Crossref PubMed Scopus (17) Google Scholar Following gene-specific amplification of CYP21, some method for detection of mutations is required. Various schemes have been used, all of which have been used for mutation detection in other genetic disorders. These include allele-specific oligonucleotide hybridization, restriction fragment length polymorphism analysis, ligase chain reaction, single-strand conformational polymorphism (SSCP), cleavase fragment length polymorphism analysis, and heteroduplex analysis. Some of these techniques can indicate only the presence of a sequence variant and must be followed by DNA sequencing for identification of a mutation. Allele-specific oligonucleotides have been used to probe PCR-amplified DNA in a dot blot format.24Rumsby G Honour JW Rodeck C Prenatal diagnosis of congenital adrenal hyperplasia by direct detection of mutations in the steroid 21-hydroxylase gene.Clin Endocrinol. 1993; 38: 421-425Crossref PubMed Scopus (17) Google Scholar In this approach, overlapping fragments of CYP21 are amplified, immobilized on a membrane and probed with labeled probes for 9 common mutations. This approach was used to identify mutations in 24 at-risk pregnancies using DNA obtained by chorionic villous biopsy.25Speiser PW White PC Dupont J Zhu D Mercado AB New MI Prenatal diagnosis of congenital adrenal hyperplasia due to 21- hydroxylase deficiency by allele-specific hybridization and Southern blot.Hum Genet. 1994; 93: 424-428Crossref PubMed Scopus (50) Google Scholar Mutations were identified in 95% of these cases, and the molecular diagnosis was clinically confirmed in 96%. Southern blot analysis was performed to detect gene deletions. The use of allele-specific PCR to detect CYP21mutations has been reported.26Wilson RC Wei JQ Cheng KC Mercado AB New MI Rapid deoxyribonucleic acid analysis by allele-specific polymerase chain reaction for detection of mutations in the steroid 21-hydroxylase gene.J Clin Endocrinol Metab. 1995; 80: 1635-1640Crossref PubMed Google Scholar In this approach, primers that are specific for eight common mutations and the corresponding primers for the wild-type sequences are used to perform PCR. Gene specificity is conferred by the use of primers for the exon 3 or exon 6 cluster of nucleotides that are characteristic of CYP21. Of 160 alleles typed using this method, mutations could be identified in 148. Deletions were detected by use of Southern blot analysis. Because absence of amplification is a possible result, it is good laboratory practice to include primers for an unrelated target to confirm the presence of both amplifiable DNA and the reagents necessary for amplification. In this approach, CYP21 is amplified with gene-specific PCR primers. Nested PCR reactions are performed using mutagenic primers to introduce restriction sites as needed.27Chung BC Hu MC Guzov VM Wu DA Structure and expression of the CYP21 (P450c21, steroid 21-hydroxylase) gene with respect to its deficiency.Endocr Res. 1995; 21: 343-352Crossref PubMed Scopus (12) Google Scholar, 28Oriola J Plensa I Machuca I Pavia C Rivera-Fillat F Rapid screening method for detecting mutations in the 21-hydroxylase gene.Clin Chem. 1997; 43: 557-561PubMed Google Scholar Restriction of the resulting PCR products is used to detect the presence of mutations. This approach has advantages and disadvantages. The use of nested PCR reactions requires additional steps and, more importantly, increases the possibility of PCR contamination because of the additional manipulations required. An advantage of this technique is that if additional restriction sites are present in the amplified product, they can serve as important internal controls of restriction enzyme activity. A few papers have reported the use of LCR for detection of CYP21 mutations. The strategy used involves initial PCR using the exon 3, 8-bp, and exon 6 cluster positions to provide targets for gene-specific priming.15Fitness J Dixit N Webster D Torresani T Pergolizzi R Speiser PW Day DJ Genotyping of CYP21, linked chromosome 6p markers, and a sex-specific gene in neonatal screening for congenital adrenal hyperplasia.J Clin Endocrinol Metab. 1999; 84: 960-966Crossref PubMed Scopus (101) Google Scholar, 22Day DJ Speiser PW White PC Barany F Detection of steroid 21-hydroxylase alleles using gene-specific PCR and a multiplexed ligation detection reaction.Genomics. 1995; 29: 152-162Crossref PubMed Scopus (109) Google Scholar A total of 4 PCR products are generated from CYP21 in overlapping segments. These PCR products are subjected to multiplex LCR using oligonucleotides specific for the mutations to be tested. By adding poly(A) tails to the LCR oligonucleotides, LCR products of different length for each mutation can be generated. Size separation of these allows for identification of the mutation present. The advantages of this approach are that much of the analysis can be multiplexed and, by using fluorescent labels on the oligonucleotides, the LCR products can be rapidly analyzed using sequencing-type apparatus. Cleavase fragment length polymorphism analysis was used to detect the presence of several frequent polymorphisms in intron 2 of CYP21.16Killeen AA Jiddou RR Sane KS Characterization of frequent polymorphisms in intron 2 of CYP21: application to analysis of segregation of CYP21 alleles.Clin Chem. 1998; 44: 2410-2415PubMed Google Scholar Subsequently, the utility of this technique for screening for common salt-wasting mutations in CYP21 was reported.29Wei WL Killeen AA Analysis of four common salt-wasting mutations in CYP21 (steroid 21- hydroxylase) by cleavase fragment length polymorphism analysis and characterization of a frequent polymorphism in intron 6.Mol Diagn. 1998; 3: 171-177Crossref PubMed Scopus (9) Google Scholar These included the intron 2 splice mutation, exon 3 8-bp deletion, exon 6 cluster, and R356W. A conclusion of the referenced study was that although the technique was able to detect the mutations, a clear distinction between homozygosity and heterozygosity was not always possible, which limits the usefulness of the method for clinical diagnostic work. SSCP analysis has been used to detect mutations in CYP21 after gene-specific PCR.30Siegel SF Hoffman EP Trucco M Molecular diagnosis of 21-hydroxylase deficiency: detection of four mutations in a single gel.Biochem Med Metab Biol. 1994; 51: 66-73Crossref PubMed Scopus (24) Google Scholar, 31Bobba A Iolascon A Giannattasio S Albrizio M Sinisi A Prisco F Schettini F Marra E Characterisation of CAH alleles with non-radioactive DNA single strand conformation polymorphism analysis of the CYP21 gene.J Med Genet. 1997; 34: 223-228Crossref PubMed Scopus (18) Google Scholar Unlike most methods, SSCP does not identify the specific mutation, and subsequent characterization is required. If samples containing known mutations are run in parallel with an unknown sample, it may be possible to infer the presence of the known mutation in the unknown sample if the SSCP conformers have identical mobility. Given the technical complexities of running SSCP gels that require rigorously controlled temperatures and possibly multiple gels involving the use of glycerol, the advantages of SSCP are not obvious, and the technique is not widely used in practice. Heteroduplex analysis has not been extensively applied to diagnosis of 21-hydroxylase deficiency, although it is a very widely used technique for mutation screening in other disorders. Heteroduplex analysis has been used to detect the T insertion mutation32Witchel SF Smith R Crivellaro CE Della Manna T Dichtchekenian V Setian N Damiani D CYP21 mutations in Brazilian patients with 21-hydroxylase deficiency.Hum Genet. 2000; 106: 414-419Crossref PubMed Scopus (12) Google Scholar and the value of heteroduplex analysis in distinguishing 2 mutations in cis versus in trans has been reported.33Bradley JF Baker D Schwartz ID Rothberg PG The importance of heteroduplexes in interpreting the results of PCR-RED diagnostic assays: application to the analysis of mutations in the steroid 21-hydroxylase gene in a case of congenital adrenal hyperplasia.Mol Diagn. 1988; 3: 119-123Crossref Scopus (9) Google Scholar As with SSCP analysis, heteroduplex analysis does not provide characterization of the mutation present; for this, sequencing or some other mutation identification scheme is necessary. Denaturing gradient gel electrophoresis of restricted genomic DNA followed by Southern blot analysis using a 21-hydroxylase probe demonstrated a striking degree of polymorphism of CYP21among normal individuals.34Reindollar RH Su BCJ Bayer SR Gray MR Rapid identification of deoxyribonucleic acid sequence differences in cytochrome P-450 21-hydroxylase (CYP21) genes with denaturing gradient gel blots.Am J Obstet Gynecol. 1992; 166: 184-191Abstract Full Text PDF PubMed Scopus (4) Google Scholar These polymorphisms were shown to be inherited as Mendelian traits and could be used to track mutant CYP21 alleles in families affected by CAH. Presumably these polymorphisms observed on denaturing gradient gel electrophoresis are due to the known polymorphisms in the exons and introns of CYP21 and CYP21P; however, a complete characterization of multiple alleles from normal individuals may reveal additional polymorphic sites. Steroid 21-hydroxylase deficiency can be treated in utero, and several papers have reported a benefit of prenatal hormonal therapy on the severity of virilization in affected newborn females.1White PC Speiser PW Congenital adrenal hyperplasia due to 21-hydroxylase deficiency.Endocr Rev. 2000; 21: 245-291Crossref PubMed Scopus (1121) Google Scholar The usual situation in which prenatal diagnosis is considered is that of a family with a previously affected child. Prenatal diagnosis of 21-hydroxylase deficiency has been available for several decades based on analysis of 17-hydroxyprogesterone in amniotic fluid. This approach is of limited value because virilization of affected females begins much earlier in pregnancy, probably around the middle of the first trimester. Prenatal hormonal therapy, which consists of administration of dexamethasone to the mother, needs to be initiated as soon as pregnancy is detected and this should not await the results of genetic testing.26Wilson RC Wei JQ Cheng KC Mercado AB New MI Rapid deoxyribonucleic acid analysis by allele-specific polymerase chain reaction for detection of mutations in the steroid 21-hydroxylase gene.J Clin Endocrinol Metab. 1995; 80: 1635-1640Crossref PubMed Google Scholar Genetic testing based on DNA analysis can be performed on chorionic villi or on amniocytes. It is very helpful to have DNA available from a previously affected child, any other children, and both parents for both mutation identification and for testing chromosomal inheritance using microsatellites. The expected fraction of fetuses that can be expected to benefit from prenatal therapy is 1/8 (ie, affected females). This treatment is not without possible risks to the mother or the 7 of every 8 fetuses who will not benefit from in utero exposure to dexamethasone.1White PC Speiser PW Congenital adrenal hyperplasia due to 21-hydroxylase deficiency.Endocr Rev. 2000; 21: 245-291Crossref PubMed Scopus (1121) Google Scholar Therapy can be stopped if genetic testing indicates the fetus is a male, or is an unaffected female.35New MI Steroid 21-hydroxylase deficiency (congenital adrenal hyperplasia).Am J Med. 1995; 98: 2S-8SAbstract Full Text PDF PubMed Scopus (64) Google Scholar In many countries and States, congenital adrenal hyperplasia is on the schedule of diseases that are tested in newborn screening programs. Interest in laboratory identification of the disease is therefore substantially greater than might be expected for a relatively uncommon disease. DNA based diagnosis has not replaced routine biochemical screening using 17-hydroxyprogesterone as the test analyte, and is unlikely to do so in the near future. Reasons for this include the relative cost of DNA testing compared with immunoassay, the complexity of DNA testing for multiple mutations, particularly the common gene deletions, and locus heterogeneity (approximately 10% of cases of CAH are due to mutations in other genes, most often steroid 11β-hydroxylase. Moreover, since ∼2% of the population are carriers, it is unclear what counseling or further testing should be offered to parents of carrier newborns, who should provide this advice, and what such interventions might involve in terms of personnel and financial costs. Of course, these considerations are not unique to CAH but are applicable to a number of other inborn errors of metabolism. In most cases, DNA analysis is performed for prenatal assessment of at-risk pregnancies or for mutation identification in affected families. In the laboratory evaluation of families with CAH, several different techniques are necessary. Southern analysis remains the most reliable method to detect gene deletions. PCR followed by one of the specific mutation detection schemes listed above is needed to identify non-deletional mutations. The choice of which mutation detection system is used depends on the laboratory; no method is clearly superior, and no PCR method is free from the potentially confounding effects of gene conversion at primer binding sites. Occasionally, DNA sequencing is needed. Amplification of overlapping regions of CYP21 is the standard method to amplify this gene. PCR of closely-linked microsatellite markers still has a role in family studies.