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Congenital adrenal hyperplasia (CAH), with an incidence rate of 1 in 10,000 to 18,000 live births, is one of the most common inherited syndromes. The condition is characterized by impaired cortisol production due to inherited defects in steroid biosynthesis. The clinical consequences of CAH, besides diminished cortisol production, depend on which enzyme is affected and whether the loss of function is partial or complete.
In >90% of CAH cases, the affected enzyme is 21-steroid hydroxylase, encoded by the CYP21A2 gene located on chromosome 6 within the highly recombinant human histocompatibility complex locus. Since sex steroid production pathways branch off proximal to this enzymatic step, affected individuals will have increased sex steroid levels, resulting in virilization of female infants. If there is some residual enzyme activity, a nonclassical phenotype results, with variable degrees of masculinization starting in later childhood or adolescence. On the other end of the severity spectrum are patients with complete loss of 21-hydroxylase function. This leads to both cortisol and mineral corticosteroid deficiency and is rapidly fatal if untreated due to loss of vascular tone and salt wasting.
Because of its high incidence rate, 21-hydroxylase deficiency is screened for in most United States newborn screening programs, typically by measuring 17-hydroxyprogesterone concentrations in blood spots by immunoassay. Confirmation by other testing strategies (eg, LC-MS/MS, CAHBS / Congenital Adrenal Hyperplasia [CAH] Newborn Screening, Blood Spot), or retesting after several weeks, is required for most positive screens because of the high false-positive rates of the immunoassays (due to physiological elevations of 17-hydroxyprogesterone in premature babies and immunoassay cross-reactivity with other steroids). In a small percentage of cases, additional testing will fail to provide a definitive diagnosis. In addition, screening strategies can miss many nonclassical cases, which may present later in childhood or adolescence and require more extensive steroid hormone profiling, including testing before and after adrenal stimulation with adrenocorticotropic hormone (ACTH)-1-24.
For these reasons, genetic diagnosis plays an important ancillary role in both classical and nonclassical cases. In addition, the high carrier frequency (approximately 1 in 50) for CYP21A2 mutations makes genetic diagnosis important for genetic counseling. Genetic testing plays a role in prenatal diagnosis of 21-hydroxylase deficiency. However, accurate genetic diagnosis continues to be a challenge because most of the mutations arise from recombination events between CYP21A2 and its highly homologous pseudogene, CYP21A1P (transcriptionally inactive). In particular, partial or complex rearrangements (with or without accompanying gene duplication events), which lead to reciprocal exchanges between gene and pseudogene, can present severe diagnostic challenges. Comprehensive genetic testing strategies must therefore allow accurate assessment of most, or all, known rearrangements and mutations, as well as unequivocal determination of whether the observed changes are located within a potentially transcriptionally active genetic segment. Testing of additional family members is often needed for clarification of genetic test results.
Ambiguous genitalia detected on prenatal ultrasound, particularly when fetus is confirmed XX female by chromosome analysis
Pregnancies at risk for 21-hydroxylase deficient congenital adrenal hyperplasia based on family history
All detected alterations will be evaluated according to American College of Medical Genetics and Genomics (ACMG) recommendations.(1) Variants will be classified based on known, predicted, or possible pathogenicity and reported with interpretive comments detailing their potential or known significance.
Because of the complexity of the genetic structure of the CYP21A2 locus, and the possibility that a patient's diagnosis may be due to other gene defects, genetic testing results should be correlated carefully with clinical and biochemical data.
This testing strategy is superior to approaches previously used, but may still miss some complex and large-scale genetic rearrangements or deletions, as well as genetic changes in intronic regions or in far upstream or downstream gene-regulatory elements that impair CYP21A2 gene expression. This can lead to false-negative test results.
Rarely, unknown polymorphisms in primer- or probe-binding sites can result in false-negative test results (DNA sequencing) or either false-positive or false-negative results (multiplex ligation-dependent probe amplification; MLPA deletion screening), due to selective allelic drop-out. False-negative or false-positive results can occur in MLPA deletion screening assays due to poor DNA quality.
In addition to disease-related probes, the multiplex ligation-dependent probe amplification technique utilizes probes localized to other chromosomal regions as internal controls. In certain circumstances, these control probes may detect other diseases or conditions for which this test was not specifically intended. Results of the control probes are not normally reported. However, in cases where clinically relevant information is identified, the ordering physician will be informed of the result and provided with recommendations for any appropriate follow-up testing.
Patients without genetic evidence for disease-causing CYP21A2 genetic changes may still have congenital adrenal hyperplasia (CAH), but due to a different enzyme defect. Additional and expanded biochemical steroid profiling is, therefore, recommended if the clinical picture is strongly suggestive of CAH.
An interpretive report will be provided.
1. Richards CS, Bale S, Bellissimo DB, et al: ACMG recommendations for standards for interpretation and reporting of sequence variations: Revisions 2007. Genet Med 2008:10(4):294-300
2. Collett-Solberg PF: Congenital adrenal hyperplasias: from clinical genetics and biochemistry to clinical practice, part I. Clin Pediatr 2001;40:1-16
3. Mercke DP, Bornstein SR, Avila NA, Chrousos GP: NIH conference: future directions in the study and management of congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Ann Intern Med 2002;136:320-334
4. Speiser PW, White PC: Medical progress: congenital adrenal hyperplasia. N Engl J Med 2003;349:776-788
5. Cradic KW, Grebe SK: A Diagnostic Sequencing Assay for CYP21 Based on Promoter Activity Provides Better Understanding of Gene Rearrangements. Abstract. Endocrine Society Annual Meeting, ENDO 2005