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Home , Aromatase

Congenital adrenal hyperplasia

Songya Pang, Lisa Ludvig Department of Pediatrics, Division of Endocrinology, University of Illinois College of Medicine, Chicago, IL 60612-7324, USA

Introduction results in adrenal crisis during the neonatal period. This form is characterized by inadequate aldoste- Until , the adrenal cortex secretes two life rone and production, reflected by low essential , and cortisol, as serum aldosterone and cortisol levels, decreased well as a clinically insignificant amount of weak excretion of these urinary metabolites, and , called dehydroepiandrosterone (DHEA). markedly increased plasma renin activity. Severe Congenital adrenal hyperplasia is a family of salt-wasting congenital adrenal hyperplasia is inherited autosomal recessive disorders of adrenal characterized by the inability of the renal tubules steroidogenesis, and is caused by deficient or to retain sodium and excrete potassium, which decreased enzymatic action essential for cortisol causes hyponatraemia and hyperkalaemia respec- . 21-hydroxylase deficiency is the tively, metabolic acidosis, and sometimes hypo- most common cause of congenital adrenal hyper- glycaemia. Adrenal crisis is manifested clinically plasia. 21-hydroxylase catalyzes the conversion of by poor feeding, lethargy, spitting up formula, progesterone to 11-deoxycorticosterone and the vomiting, loose stools or diarrhea, weak cry, conversion of 17-hydroxyprogesterone (17-OHP) dehydration, hypotension, weight loss, and failure to 11-deoxycortisol (Fig. 1). 21-hydroxylase defi- to thrive. In addition, the random basal serum 17- ciency results in cortisol deficiency with or OHP level is generally highly elevated. without concomitant aldosterone deficiency. Cor- The severe classic non-salt-wasting or simple tisol deficiency results in hyperplastic changes in virilizing form is diagnosed when the affected the adrenals due to increased adrenocortical tropic newborn or child demonstrates neither clinical nor (ACTH) secretion, which results in biochemical signs or symptoms of salt-wasting. increased 17-OHP and secretion from This form is characterized by retaining adequate early foetal life. Aldosterone deficiency leads to aldosterone secretion, normal renin and aldoste- salt wasting, dehydration, and can eventually lead rone dynamics, as well as intact renal sodium to adrenal shock in young infants. 21-hydroxylase conservation. The symptoms include clitoral or deficiency results in a wide range of clinical mani- penile enlargement, accelerated growth rate with festations due to the varying degrees of marked age advancement, premature sexual insufficiency or deficiency. hair growth, and eventual early growth cessation resulting in short stature. Random basal serum 17- OHP levels are generally highly elevated in this Clinical and biochemical manifestations form as well. The mild or late onset form is characterized by This wide clinical spectrum of 21-hydroxylase mild androgen excess. The signs of this condition deficiency ranges from the most severe salt-was- include mild clitoral enlargement, mildly accele- ting classic form to the mild non-classic form [1- rated growth with eventual bone age advance- 3]. The most severe salt-wasting classic form ment, premature pubarche, acne in young chil-

Annales Nestlé 1998;56:101-108 101 Songya Pang, Lisa Ludvig

Cholesterol

Mineralocorticoids Glucocorticoids Sex Dehydroepiandrosterone sulfate 17α-hydroxylase 17, 20 17β-HSD 17-OH pregnenolone Dehydroepi- Androstenediol androsterone

3β-HSD 3β-HSD 3β-HSD 3β-HSD

17α-hydroxylase 17, 20 lyase 17β-HSD Progesterone 17-OH progesterone ACTH

21-hydroxylase 21-hydroxylase Aromatase Aromatase

17β-HSD Deoxycorticosterone 11-deoxycortisol 11β-hydroxylase 11β-hydroxylase Corticosterone Cortisol 18-hydroxylase 18-OH corticosterone 18-OH dehydrogenase Aldosterone

Figure 1: Schematic of adrenal steroidogenesis (Pang and Shook [38], used with permission).

dren, and hirsutism and menstrual disorders in of congenital adrenal hyperplasia patients [6]. older females. Occasionally, these patients have Two human P450 21-hydroxylase short stature and sometimes post-pubertal affected that include the active functional P450c21B males may be infertile. These patients invariably or CYP21B , and the inactive P450c21A have elevated ACTH-stimulated 17-OHP levels, or CYP21A pseudogene, were identified. Both greater than are levels found in the carriers for 21- CYP21-hydroxylase genes consist of 10 and hydroxylase deficiency. 9 introns, and are 98% homologous in genomic sequences [4, 5]. The CYP21A pseudogene differs from the active CYP21B gene by 11 deleterious Molecular basis of 21-hydroxylase deficiency point mutations located in the , intron, and regions of the gene. These point muta- The genes encoding 21-hydroxylase are in the tions abolish 21-hydroxylase activity [4, 5]. Dele- class III region of the HLA complex on the short terious mutations in the CYP21B gene [7, 9, 11, arm of 6 [4, 5]. Because of the gene- 12, 14, 19] reported in 307 patients from 5 popula- tic linkage between the HLA class I and II genes tions including France, Japan, Sweden, New and 21-hydroxylase genes, the prediction of the York, and Texas, revealed a large 21B gene dele- genotype is possible by HLA typing in families tion and large 21B to A gene conversion muta-

102 Annales Nestlé 1998;56:101-108 Congenital adrenal hyperplasia

Figure 2: A. CYP21B and CYP21A gene mapping within the HLA complex. B: Schematic of 21B gene structure and reported mutations from the alleles of patients with severe salt wasting (SW), moderately severe simple virilizing (SV), and mild non-clas- sic (NC) 21-hydroxylase deficiency. • CYP21A gene sequence; numbers in the mutant allele boxes indicate codon numbers; num- bers in the shaded boxes indicate exon numbers. (Modified from Pang S [2], used with permission).

Annales Nestlé 1998;56:101-108 103 Songya Pang, Lisa Ludvig tions in 32% of alleles in salt-wasting patients. Neonatal screening for Fifty-six percent of alleles in salt-wasters had an 21-hydroxylase deficiency intron 2 point mutation that affects the RNA spli- cing mechanism. Other point mutations found in The goal of newborn screening for congenital the salt-wasting patients are primarily frameshifts, adrenal hyperplasia is to prevent life-threatening multiple amino substitutions, and premature stop adrenal crisis, thereby averting shock and its codon mutations (Fig. 2). sequel (i.e. damage), and death of affected In simple virilizing patients, two common newborns, as well as the prevention of male sex alleles were a codon 172 substitution mutation assignment in virilized female newborns, and the and the intron 2 point mutation [14, 19, 20]. In late prevention of the progressive effects of excess onset non-classic patients the most common allele adrenal androgens that ultimately cause short sta- was a codon 281-substitution mutation [11, 12, ture, gender confusion in girls, and psychosexual 14, 19, 21-24]. Of 13 point mutations proven to be disturbances in boys and girls. Therefore, new- deleterious, 10 were 21A gene sequences, indica- born screening for congenital adrenal hyperplasia ting a micro-sequence transfer from the A to B is aimed at detecting newborns affected with clas- gene during meiosis by a poorly understood gene sic 21-hydroxylase deficiency. conversion process. Newborn screening for congenital adrenal In order to determine the relationship between hyperplasia first became possible in 1977 using a the genotypes and phenotypes in 21-hydroxylase heel stick capillary blood sample specimen impre- deficiency in a greater patient population, due to gnated on filter paper [25]. The feasibility of the divergent reports in this regard in smaller patient newborn screening was first demonstrated by the populations [14-24], we compared the reported in Alaskan pilot programme [26]. Since then, nume- vitro mutant gene activity to the reported clinical rous newborn screening programmes have been form of 21-hydroxylase deficiency in all reported initiated in as many as 15 countries, including patient populations [2, 3]. Compared to the acti- Brazil, Canada, France, Germany, Israel, Italy, Ja- vity of a single wild type allele, the mutant geno- pan, New Zealand, Portugal, Saudi Arabia, Scot- type alleles were grouped according to the repor- land, Spain, Sweden, Switzerland, and the United ted degree of in vitro activity. Two wild type States. They are currently in effect in 13 countries alleles were estimated to exhibit 100% enzyme and, in the Unites States alone, including Ala- activity. The combined mutant allele activity was bama, Alaska, Florida, Georgia, Hawaii, Illinois, compared to the clinical forms. In salt-wasting Iowa, Michigan, Minnesota, New England, North patients, 94% of the patients had 0% activity Carolina, North Dakota, Pennsylvania, South genotypes, but 6% of the patients had ≥1-2% acti- Carolina, Texas, Washington, and Wisconsin, 17 vity genotypes. In simple virilizing patients, 79% regional programmes are in progress. To date, of the patients had ≥1-2% activity genotypes, but greater than 10 million newborns have been scree- 21% had near 0% activity genotypes. In late-onset ned for congenital adrenal hyperplasia in 13 coun- patients, 92% had ≥1 –2% activity genotypes, but tries between 1978 and 1995. The details of this 8% had near 0% activity genotypes. The geno- newborn screening update, with reports on the types and phenotypes agreed in 89% of the total disease incidence and phenotypic variability and patient population reported, but did not agree in the effectiveness of screening, are currently under the remaining 11% [2, 3]. This lack of correlation investigation by an international consortium. may be due to insufficient or inaccurate genoty- ping techniques in some laboratories, variable dia- gnostic criteria used for the clinical forms, envi- Newborn screening procedures ronmental factors such as the presence or absence Optimal screening for congenital adrenal hyper- of stress, and constitutional factors contributing to plasia requires early sample collection, immediate phenotypic differences even among some sibling and reliable analysis of 17-OHP levels, optimal patients. Other poorly understood mechanisms 17-OHP cut-off levels to distinguish between also have been proposed. affected and unaffected newborns, prompt and

104 Annales Nestlé 1998;56:101-108 Congenital adrenal hyperplasia clear communication of suspected results to the most programmes or as based on the laboratory health care professional and to family members, experience with normal newborns [27]. Theoreti- diagnostic confirmation of suspected newborns, cally newborn screening 17-OHP concentrations and report of false-positive and false-negative among laboratories should be comparable regard- results to the screening programme. less of the assay method, however, there is a A filter paper blood spot sample is used for considerable variation in cut-off levels among the congenital adrenal hyperplasia screening during programmes. This can be attributed to different the concurrent screening test for other disorders. reagents used in diverse assay systems, the Therefore, other disorder screening influences the varying thickness and density of the filter paper age at which congenital adrenal hyperplasia scree- used for sample collection, and the background of ning samples are collected. The majority of new- the reference newborns (birth weight, gestational born screening samples for congenital adrenal age and sample age) selected to establish normal hyperplasia are collected between 3 to 5 days of levels in full term and preterm infants. life [27], although delayed turnaround times were noted in some programmes [29]. Almost all of the screening programmes worldwide employ a Screening test reliability single sample-screening test. This single sample The majority of false-positive results have been screening method offers the advantage of expedi- due to low birth weight and premature birth. 17- ted results and a less expensive service, but may OHP levels are generally higher in preterm infants, entail a slightly greater chance of inaccurate suggesting that some factor such as the inability to results in borderline cases. A small number of pro- metabolize 17-OHP or illness related stress might grammes perform a second test of the initial cause elevated 17-OHP levels in these newborns. sample to confirm borderline cases from the first Therefore, separate normative reference levels screening [27]. Testing of both initial and second have been established based on birth weight or filter paper screening samples provides greater gestational age to minimize high false-positive accuracy, however, it is more expensive and rates among low-birth-weight or preterm new- medical action may be necessary based on the borns. This measure has minimized unnecessary results of the first test, because the second sample recall and medical work-up of unaffected cases, testing is time consuming. A few programmes which is costly. A recent study compared the worldwide mandate two sample screenings [27, false-positive rate of newborn screening per case, 30]. The second sample screening method mini- as based on the three-level weight-adjusted 17- mizes both false-positive and false-negative OHP cut-off levels in normal and low-birth-weight results by detecting newborns affected by the full newborns, to the false-positive rate of screening spectrum of congenital adrenal hyperplasia, i.e. by two-level weight-adjusted 17-OHP cut-off from severe to mild cases. levels. This reduced the false-positive rate signifi- cantly in all infants [31]. Many programmes with Screening assay methods acceptable false-positive results are using two- level weight-adjusted 17-OHP cut-off levels [27]. The assay techniques utilized for the initial scree- Ongoing modification of the test, cut-off levels, ning of congenital adrenal hyperplasia in neonates and procedures are nevertheless necessary to include radioimmunoassay, enzyme-linked immu- minimize false-positive rates in all programmes. nosorbent assay (ELISA), and time-resolved flu- The false-negative rate for screening is low [27], roimmunoassay. These assays measure the 17- but requires further study to determine the true OHP concentration in the eluate of a filter paper false-negative rate for non-salt-wasting forms. blood spot sample obtained by the heel prick tech- Based on the report of greater than 7.5 million nique. The 17-OHP cut-off levels for determining newborns screened for congenital adrenal hyper- positive cases via screening for congenital adrenal plasia [27, 32-37], the incidence of the severe hyperplasia have been established at greater than classic forms among the homogenous or heteroge- the 97-99th percentile of the normal mean level in neous general populations ranges from as high as

Annales Nestlé 1998;56:101-108 105 Songya Pang, Lisa Ludvig

1:5000 live births in Saudi Arabia to as low as evaluating false-positive cases and the undesi- 1:23,000 live births in New Zealand. The inci- rable psychological impact on the families, conti- dence of congenital adrenal hyperplasia in North nue to be the major pitfalls of congenital adrenal America was 1:15,000 live births, and in Europe, hyperplasia screening. As a result of screening, incidences generally ranged between 1:11,000 the number of newborns suspected to have conge- and 1:15,000 live births, with the exception of nital adrenal hyperplasia increased, and new gui- Scotland, Spain, and the combined regions of delines became necessary for evaluating new- Italy, where the incidences were slightly lower borns with positive screening results. [27, 32-34]. The incidence in Japan was reported The costs of the screening can be considered to be approximately 1: 19,000 live births. Neona- acceptably low or high depending on the goal of tal screening for 21-hydroxylase deficiency revea- the programme [30, 38]. However, the lifetime tax led a higher incidence of the disease in almost all- contribution to society of a productive subject screening regions compared with the findings of with congenital adrenal hyperplasia, as discussed case surveys by clinical ascertainment [27]. previously [27], may outweigh the cost of detec- ting one case, and should be considered in the cost-benefit analysis of newborn screening for Benefits of congenital adrenal hyperplasia congenital adrenal hyperplasia. screening Early diagnosis of congenital adrenal hyperplasia through screening benefited 70% of the affected Conclusions newborns with classic 21-hydroxylase deficiency who were not clinically suspected [27]. In almost In general, two-level weight-adjusted 17-OHP cut- all screened populations, adrenal crisis has been off levels in almost all programmes have been averted in the majority of newborns with the established to guide the evaluation of full-term severe salt-wasting forms, and prevention of life- and preterm newborns with exceptionally eleva- time incorrect male gender assignment in severely ted (urgent) or moderately elevated (suspected) virilized females has been obtained. Thus, scree- levels of 17-OHP. Immediate medical evaluation ning has helped to prevent the morbidity of is necessary in newborns with ambiguous genita- delayed diagnosis in these cases [27]. lia with urgent or suspected levels, in sick male newborns with urgent or suspected levels, and in Pitfalls and cost of congenital adrenal sick females with urgent levels. Although evalua- hyperplasia screening tion for 21-hydroxylase deficiency is necessary in clinically normal female newborns with urgent Neonatal screening, however, was not sufficient levels and in clinically normal male and sick for preventing adrenal crisis in a few cases due to females with suspected levels, these newborns are delayed screening turnaround time, human error at low risk of salt wasting crisis. in the screening procedure, or lack of understan- ding of the implication of a positive screening results among health care professionals [27]. References These pitfalls of newborn screening have been diminishing as screening programmes strive for 1. Miller WL, Levine LS. Molecular and clinical advances greater efficacy and to educate health professio- in congenital adrenal hyperplasia. J Pediatr 1987;111:1-7. 2. Pang S. Congenital adrenal hyperplasia. In: Rosenfield nals in regard to management of congenital adre- R, ed. Baillière’s clinical obstetrics and gynecology: nal hyperplasia. The false-positive rate in preterm hyperandrogenic states in hirsutism. London: Baillière low-birth-weight infants, which was initially high, Tindal, 1997:11:281-306. also has been improving. This has improved by 3. Pang S. Congenital adrenal hyperplasia. In: Endocrino- logy and , Clinics of North America, 1998; developing adequate reference 17-OHP levels in 26: 853-91. preterm infants [27, 31]. Nevertheless, issues rela- 4. White PC, Grossberger D, Onufer BJ, et al. Two genes ting to false-positive results, including the cost of encoding steroid 21-hydroxylase are located near the

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genes encoding the fourth component of complement in 19 Wedell A, Thilen A, Ritzen EM, et al. Mutational spec- man. Proc Natl Acad Sci USA 1985;82:1089-93. trum of the steroid 21-hydroxylase gene in Sweden: 5. Chang S, Chung B. Difference in transcriptional activity Implications for genetic diagnosis and association with of two homologous CYP21A genes. Mol Endocrinol disease manifestation. J Clin Endocrinol Metab 1994; 1995;9:1330-6. 78:1145-52. 6. Levine LS, Zachmann M, New MI, et al. Genetic map- 20. Chiou S, Hu M, Chung B. A missense mutation at Ile ping of the 21-hydroxylase deficiency gene within the 172→Asn or Arg 356→Trp causes steroid 21-hydroxy- HLA linkage group. N Engl J Med 1978;299:911-5. lase deficiency. J Biol Chem 1990; 265:3549-52. 7. Higashi Y, Hiromasa T, Tanae A, et al. Effects of indivi- 21. Helmberg A, Tusie-Luna M, Tabarelli M, et al. R339H dual mutations in the P-450 (C21) pseudogene on the and P453S: CYP21 mutations associated with non-clas- P-450 (C21) activity and their distribution in the patient sic steroid 21-hydroxylase deficiency that are not appa- genomes of congenital steroid 21-hydroxylase defi- rent gene conversions. Mol Endocrinol 1992;6:1318-22. ciency. J Biochem 1991;109:638-44. 22. Owerbach D, Sherman L, Ballard A, Azziz R. Pro-453 to Ser mutation in CYP21 is associated with non classic ste- 8. Higashi Y, Tanae A, Inoue H, et al. Aberrant splicing roid 21-hydroxylase deficiency. Mol Endocrinol 1992;6: and missense mutations cause steroid 21-hydroxylase [P- 1211-5. 450 (C21)] deficiency in humans: Possible gene conver- 23. Tardy V, Carel JC, Forest MG, et al. Non-classic forms sion products. Proc Natl Acad Sci USA 1988;85:7486- of 21-hydroxylase deficiency revisited by molecular bio- 90. logy. In: 10th International Congress of Endocrinology 9. Higashi Y, Tanae A, Inoue H, et al. Evidence for fre- Program & Abstracts, San Francisco, CA 1996, P2-737. quent gene conversion in the steroid 21-hydroxylase 24. Tusie-Luna M, Speiser PW, Dumic M, et al. A mutation p-450 (C21) gene: implications for steroid 21-hydroxy- (Pro-30 to Leu) in CYP21 represents a potential non- lase deficiency. Am J Hum Genet 1988;42:17-25. classic steroid 21-hydroxylase deficiency allele. Mol 10. Morel Y, Miller W. Clinical and molecular genetics of Endocrinol 1991;5:685-92. congenital adrenal hyperplasia due to 21-hydroxylase 25. Pang S, Hotchkiss J, Drash AL, Levine LS, New M. deficiency. Adv Hum Genet 1991:20:1-68. Microfilter paper method for 17 α-hydroxyprogesterone 11. Mornet E, Crete P, Kuttenn F, et al. Distribution of dele- radioimmunoassay: its application for rapid screening for tions and seven point mutations on CYP21B genes in congenital adrenal hyperplasia. J Clin Endocrinol Metab three clinical forms of steroid 21-hydroxylase defi- 1977;45:1003-8. ciency. Am J Hum Genet 1991;48:79-88. 26. Pang S, Murphey W, Levine LS, et al. A pilot newborn 12. Owerbach D, Ballard A, Drazin MB. Salt-wasting conge- screening for congenital adrenal hyperplasia in Alaska. J nital adrenal hyperplasia: Detection and characterization Clin Endocrinol Metab 1982;55:413-20. of mutations in the steroid 21-hydroxylase gene, CYP21, 27. Pang S, Clark A. Congenital adrenal hyperplasia due to using the polymerase chain reaction. J Clin Endocrinol 21-hydroxylase deficiency: newborn screening and its Metab 1992;74:553-8. relationship to the diagnosis and treatment of the disor- 13. Speiser PW, Agdere L, Ueshiba H, et al. Aldosterone der. Screening 1993;2:105-39. synthesis in patients with salt-wasting congenital adrenal 28. Pang S, Wallace MA, Hofman L, et al. Worldwide expe- hyperplasia (21-hydroxylase deficiency) and complete rience in newborn screening for classical congenital absence of adrenal 21-hydroxylase (p450c21) N Engl J adrenal hyperplasia due to 21-hydroxylase deficiency. Med 1991;324:145-9. Pediatrics 1988;81:866-74. 14. Speiser PW, Dupont J, Zhu D, et al. Disease expression 29. Listernick R, Frisone L, Silverman BL. Delayed diagno- and molecular genotype in congenital adrenal hyperpla- sis of infants with abnormal neonatal screens. JAMA sia due to 21-hydroxylase deficiency. J Clin Invest 1992; 1992;267:1095-9. 90:584-95. 30. Brosnan CA, Riley WJ, Brosnan PG, Therrell BL. The cost-effectiveness of newborn screening for congenital 15. Tusie-Luna M, Traktman P, White PC. Determination of adrenal hyperplasia (CAH) due to 21-hydroxylase defi- functional effects of mutations in the steroid 21-hydroxy- ciency. In: Proceeding, Third Meeting of the Internatio- lase gene (CYP21) using recombinant vaccinia virus. J nal Newborn Screening Meeting, Jamaica Plain, MA. Biol Chem 1990;265:20916-22. New England Regional Newborn Screening Program; 16. Wedell A. Molecular approaches for the diagnosis of 21- 1996:85. hydroxylase deficiency and congenital adrenal hyperpla- 31. Allen DB, Hoffman GL, Mahy SL, et al. Improved preci- sia. Clin Lab Med 1996;16:125-37. sion of newborn screening for congenital adrenal hyper- 17. Wedell A, Luthman H. Steroid 21-hydroxylase defi- plasia using weight adjusted criteria for 17-hydroxypro- ciency: two additional mutations in salt-wasting disease gesterone levels. J Pediatr 1997, 130:120-33. and rapid screening of disease-causing mutations. Hum 32. Balsamo A, Cacciari E, Piazzi S, et al. Congenital adre- Mol Genet 1993;2:499-504. nal hyperplasia: neonatal mass screening compared with 18. Wedell A, Ritzen ME, Haglund-Stengler B, et al. Steroid clinical diagnosis only in the Emilia-Romagna region of 21-hydroxylase deficiency: three additional mutated Italy, 1980-1995. Pediatrics 1996;98:362-7. alleles and establishments of phenotype-genotype rela- 33. Cutfield WS, Webster D. Newborn screening for conge- tionships of common mutations. Proc Natl Acad Sci USA nital adrenal hyperplasia in New Zealand. J Pediatr 1992;89:7232-6. 1995;126:118-21.

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34. Dotti G, Pagliardini S, Vuolo A, et al. Congenital adrenal 21-hydroxylase deficiency in Israel. In: Programs and hyperplasia in the experience of the Piemonte and Valle Abstracts of the 3rd International Newborn Screening d’Aosta regions program (1987-1985). In: Programs and Meeting, Jamaica Plain, MA. New England Regional Abstracts of the 3rd International Newborn Screening Newborn Screening Program; 1996;87-8. Meeting, Boston:1996:82-5. 37. Al-Nuaim AA. Newborn screening program (NSP) in 35. Nordenstrom A, Thilen A, Hagenfeldt L, et al. Benefits Saudi Arabia (SA). In: Programs and Abstracts of the 3rd of screening for congenital adrenal hyperplasia (CAH) in International Newborn Screening Meeting, Jamaica Sweden. In: Proceedings, Third Meeting of the Interna- Plain, MA. New England Regional Newborn Screening tional Society for Neonatal Screening. Levy HL, Hermos Program;1996:89. RJ, Grady GF, eds. Jamaica Plain, MA. New England 38. Pang S, Shook M. Current status of neonatal screening Regional Newborn Screening Program;1996:211-6. for congenital adrenal hyperplasia. Curr Opin Pediatr 36. Sack J, Front H, Kaiserman I, Schreiber M. Screening for 1997:419-23.

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