Prenatal Diagnosis Of 21-hydroxylase Deficiency Research Articles

Noninvasive prenatal diagnosis of 21-hydroxylase deficiency using target capture sequencing of maternal plasma DNA

Noninvasive prenatal diagnosis of 21-hydroxylase deficiency using target capture sequencing of maternal plasma DNA

Genetic analysis and prenatal diagnosis of 21-hydroxylase deficiency by Sanger sequencing combined with MLPA

Genetic analysis and prenatal diagnosis of 21-hydroxylase deficiency by Sanger sequencing combined with MLPA

Noninvasive prenatal diagnosis of 21-Hydroxylase deficiency using target capture sequencing of maternal plasma DNA

Here, we aimed to validate a noninvasive method using capture sequencing for prenatal diagnosis of congenital adrenal hyperplasia due to 21-Hydroxylase deficiency (21-OHD). Noninvasive prenatal diagnosis (NIPD) of 21-OHD was based on 14 plasma samples collected from 12 families, including four plasma sample collected during the first trimester. Targeted capture sequencing was performed using genomic DNA from the parents and child trios to determine the pathogenic and wild-type alleles associated with the haplotypes. Maternal plasma DNA was also sequenced to determine the fetal inheritance of the allele using hidden Markov model-based haplotype linkage analysis. The effect of fetal DNA fraction and sequencing depth on the accuracy of NIPD was investigated. The lower limit of fetal DNA fraction was 2% and the threshold mean sequence depth was 38, suggesting potential advantage if used in early gestation. The CYP21A2 genotype of the fetus was accurately determined in all the 14 plasma samples as early as day 1 and 8 weeks of gestation. Results suggest the accuracy and feasibility of NIPD of 21-OHD using a small target capture region with a low threshold for fetal DNA fraction and sequence depth. Our method is cost-effective and suggests diagnostic applications in clinical practice.

Carrier analysis and prenatal diagnosis of congenital adrenal hyperplasia caused by 21-hydroxylase deficiency in Chinese.

Congenital adrenal hyperplasia (CAH) is a common autosomal recessive disorder mainly caused by defects in the steroid 21-hydroxylase (CYP21) gene. We screened 1,000 healthy people, using a previously developed differential PCR method combined with single-strand conformation polymorphism and amplification-created restriction site methods for the carrier detection of the CYP21 gene deficiency. Our results indicated that the rate of occurrence of the heterozygous CAH carrier was about 12 in 1,000, with a gene frequency of 0.0060 and an incidence frequency of 1 in 28,000 in the Chinese population. In addition, 9 cases of CAH families were performed with prenatal diagnosis. Among them, 3 cases were diagnosed as the severe form, 4 cases carried the heterozygous mutation, and 2 were normal. This is the first report of carrier frequency analysis and prenatal diagnosis of 21-hydroxylase deficiency in Chinese.

Profiling steroid hormones in amniotic fluid of midpregnancy by routine stable isotope dilution/gas chromatography-mass spectrometry: reference values and concentrations in fetuses at risk for 21-hydroxylase deficiency.

Using routine stable isotope dilution/gas chromatography-mass spectrometry, 17-hydroxyprogesterone, androstenedione, testosterone, dehydroepiandrosterone, androstanediol, and 5alpha-dihydrotestosterone have been profiled in amniotic fluid of midgestation in 77 normal fetuses and 38 untreated or dexamethasone-treated fetuses at risk for 21-hydroxylase deficiency. Dexamethasone was suspended 5-7 days before amniocentesis. In normal fetuses, amniotic fluid concentrations (median, range; nanograms per mL) of 17-hydroxyprogesterone did not reveal a sex difference (1.48, 0.21-4.96), whereas those of androstenedione were lower in females (0.53, 0.00-2.71) than in males (0.93, 0.29-1.98). Testosterone levels were higher in males (0.24, 0.00-0.50) than in females (0.00, 0.00-0.27). No sex difference was found for dehydroepiandrosterone (0.47, 0.19-1.77). Levels of androstanediol and 5alpha-dihydrotestosterone were below the detection limit of our method in most cases. Regarding prenatal diagnosis of 21-hydroxylase deficiency, 17-hydroxyprogesterone and androstenedione presented the diagnostically most valuable steroids and were of equal diagnostic potential. They permitted successful diagnosis in 36 of 37 fetuses at risk: 12 were untreated and unaffected, 13 were treated and unaffected, 4 were untreated and affected (3 salt wasters and 1 simple virilizer), and 8 were treated and affected (5 salt wasters and 3 simple virilizers). In the latter group, one simple virilizer revealed normal steroid concentrations. Isotope dilution/gas chromatography-mass spectrometry, providing the highest specificity in steroid analysis, is proposed for routine use in clinical steroid analysis whenever maximal reliability is requested. Our study provides the first mass spectrometric reference data on amniotic fluid steroid concentrations and underscores the high accuracy of prenatal hormonal diagnosis of 21-hydroxylase deficiency.

Congenital adrenal hyperplasia in pregnancy

The congenital adrenal hyperplasias (CAH) are a group of inherited enzymatic defects of adrenal steroid biosynthesis. Deficiencies of each enzyme required in the steroid biosynthesis pathway are known, and these deficiencies are all inherited as autosomal recessive disorders. During pregnancy, maternal and fetal problems are confined to women who have 21-hydroxylase deficiency (P450c21 deficiency), 11-hydroxylase deficiency (P450c11 deficiency), and 3 beta-hydroxysteroid dehydrogenase deficiency (3 beta HSD deficiency), because other adrenal enzyme deficiencies are not compatible with fertility. The interposition of the placenta on the hypothalamic-pituitary-adrenal axis and other endocrine changes during pregnancy impact considerably on the clinical evaluation of the congenital adrenal hyperplasias. Successful management of CAH in pregnancy requires a firm knowledge of normal adrenal anatomic and endocrine changes that occur during gestation. Women with severe forms of CAH have decreased fertility rates because of oligo-ovulation, and successful conception requires a combination of good therapeutic compliance, careful endocrine monitoring, and often ovulation induction. From a fetal and neonatal standpoint, accurate prenatal diagnosis of 21-hydroxylase deficiency and 11-hydroxylase deficiency is now possible, which allows for prenatal treatment in an attempt to minimize clinical problems in the neonates. Prevention of masculinization of affected female fetuses by corticosteroid suppression has been attempted in both 21-hydroxylase deficiency and 11beta-hydroxylase deficiency CAH, with variable degrees of success. This review provides an overview of the congenital adrenal hyperplasias and their management during pregnancy.

Prenatal diagnosis and treatment of 21-hydroxylase deficiency

Prenatal diagnosis of 21-hydroxylase deficiency, the most common use of congenital adrenal hyperplasia (CAH), has benefited from the advances in endocrinologic and molecular genetic studies. In 1976, prenatal diagnosis of the disease was first attempted by measuring 17-hydroxyprogesterone in the amniotic fluid in the second trimester of pregnancy. Discovery of a close linkage between HLA and the disease gave a second approach for prenatal diagnosis, the latter being made by linkage study of the haplotypes of the index case in a given family. Diagnosis was later made directly by molecular biology. Currently, the studies of the C4-CYP21B gene locus by Southern blotting and the CYP21B gene mutations by PCR ethods simplify the diagnositic procedure of an early and accurate prenatal diagnosis in the first trimester. In these conditions all families are now informative. Moreover, using a direct genetic analysis associated with the possibility of detecting the heterozygotes in a non-related CAH population, a prenatal diagnosis can be done in a family without a previously CAH affected child. From our results in a series of 274 pregnancies at risk for CAH in whom prenatal diagnosis has been made by these different approaches, it can be cncluded that steroid analysis in the amniotic fluid is an accurate method but provides only a late (second trimester) diagnosis, while an early and accurate diagnosis now relies on adequate molecular enetic studies on chorion villus biopsies. In the aim to prevent the virilization of the external genitalia in CAH female fetuses, prenatal treatment was instituted in our group in 1979 by giving dexamethasone to the mother. This prenatal treatment appears safe for the fetus and the child and is effective in preventing virilization of CAH affected females. Although the degree of prevention is not always complete in all cases, the advantages of prenatal treatment are prevailing over the complications observed in a few mothers.

Problems in diagnosis and management of congenital adrenal hyperplasia due to 21-hydroxylase deficiency

A number of biochemical tests have been utilized to assist the diagnosis of steroid 21-hydroxylase deficiency. The specificity and accuracy of plasma 17-hydroxyprogesterone assays are important. A profile of steroids in urine by gas chromatography and mass spectrometry is the definitive test. Molecular biology is not practical for the diagnosis of a new case. The ACTH stimulation test for detection of heterozygotes is a poor discriminant. Fertility in patients with congenital adrenal hyperplasia may be due to excess of progesterone as well as of androgens. Gene amplification offers the best approach in molecular biology for the prenatal diagnosis of 21-hydroxylase deficiency.

Highly polymorphic XbaI RFLPs of the human 21-hydroxylase genes among Chinese.

A highly polymorphic XbaI restriction site flanking the human 21-hydroxylase genes (21-OHA and 21-OHB) was found with the probe pC21/3c, the cDNA of the 21-hydroxylase gene. From the results of RFLP analysis of 10 subjects with congenital adrenal hyperplasia (CAH) owing to 21-hydroxylase deficiency and their parents, at least four polymorphic fragments (30 kb, 27 kb, 25 kb, and 15 kb) resulting from cleavage at the polymorphic endonuclease sites outside the genes were found, and at least 10 different polymorphic patterns were observed among Chinese. These results indicate that these polymorphic loci are very informative for prenatal diagnosis of 21-hydroxylase deficiency.

Prenatal diagnosis/treatment in families at risk for infants with steroid 21-hydroxylase deficiency (congenital adrenal hyperplasia)

The most common enzymatic defect of steroid synthesis is adrenal steroid 21-hydroxylase deficiency. Inhibited formation of cortisol causes increased pituitary release of ACTH, driving the adrenal cortex to overproduce androgens, whose synthesis does not involve the 21-hydroxylase enzyme. This hormonal setting is established in the embryonic period and affects development of genetic females, misdirecting differentiation of the external genitalia toward male type. At birth, the genitalia are visibly ambiguous (enlarged clitoris, fused labia) or in some cases even male in appearance {phallus with urethral opening, rugated scrotal sac), leading to wrong sex assignment. Adrenal steroid 21-hydroxylase deficiency is the most common basis of female pseudohermaphroditism. These females, however, have normal fertility and potential for gestation (gonads are functional and the internal duct-derived structures are well-formed), thus the sex of rearing should always be female. Management is by life-long hormonal (glucocorticoid) replacement, with surgical correction of the genital ambiguity. Prenatal diagnosis of 21-hydroxylase deficiency, first possible by steroid assay of the amniotic fluid, has utilized HLA typing for identification of loci (antigens B and DR) in close linkage with the 21-hydroxylase gene, and now increasingly relies on DNA analysis for linked HLA or C4 genes or for mutant 21-hydroxylase alleles directly by molecular genetic techniques. The most recent clinical advance is a program of combined prenatal diagnosis with karyotyping and suppression of fetal androgen production in genetic females by steroid administration to the mother. This is the first instance of an inborn metabolic error to be prenatally treated. A series of 85 managed pregnancies is reported on, including accuracy of diagnosis, response of the mother to steroid treatment, and outcome for treated and untreated male and female fetuses (of 77 born by 6/91). Prenatal diagnosis by current techniques is accurate. Normal growth and development patterns postnatally suggest that dexamethasone treatment is safe.

Prenatal diagnosis of 21-hydroxylase deficiency by rflp analysis of the 21-hydroxylase, complement C4, and HLA class II genes

In 19 pregnancies at risk for 21-hydroxylase deficiency (21OHD) in 18 families with at least one affected child, prenatal diagnosis was performed by RFLP analysis using the enzymes Taq I and EcoRI and the DNA probes specific for the 21OH genes, the closely linked complement C4 genes and the highly polymorphic HLA class II genes DRB, DQB, and DPB. For fetal DNA analysis either chorionic villi or cultivated amniotic cells were used. In all 19 cases, a clear prenatal diagnosis was possible either with the 21OH probe alone or in most cases, by combining the results of the different closely linked loci.

A Case of First Trimester Prenatal Diagnosis of 21-Hydroxylase Deficiency with Human Complement C4 cDNA Probe.

Early prenatal diagnosis of 21-hydroxylase (21-OHase) deficiency would enable treatment to be done to protect the fetus from masculinization and/or life-threatening adrenal crisis at birth. We report here the prenatal diagnosis of 21-OHase deficiency with human complement component C4 cDNA to probe DNA from chorionic villi at 10 weeks of gestation. Southern analysis with human C4 cDNA identified TaqI restriction fragment length polymorphisms (RFLPs) in the family. Family analysis with these RELPs showed that the fetus was not affected at greater than 99% probability, because the frequency of recombination between the 21-OHase B gene and the C4 gene would be extremely low.

Prenatal DNA analysis in four embryos/fetuses at risk of 21-hydroxylase deficiency.

Prenatal diagnosis of 21-hydroxylase deficiency (21-OHD) in two unrelated embryos and two fetuses was attempted with the Southern hybridization method using the 21-hydroxylase (21-OHase) complementary DNA as a probe. The two embryos whose genomic DNA was extracted from their chorionic villi both had four TaqI fragments (3.7 kb, 3.2 kb, 2.4 kb and 2.3 kb) identical to those of their respective parents and normal controls, while the DNA from each proband of these two families lacked with the 3.7 kb and the 2.3 kb fragments corresponding to the functional 21-OHase gene (21-OHase B gene). These findings indicated that none of the embryos examined were deletion homozygotes for the 21-OHase B gene. In the two fetuses, only amniotic fluid cells were available for prenatal diagnosis. The results of Southern hybridization analysis were uninformative since all family members, including the probands and fetuses, had all four TaqI fragments. Linkage studies between 21-OHD and human leukocyte antigen (HLA) haplotypes and those between the disease and restriction fragment length polymorphisms of the 4th complement gene revealed that the fetus of one family was normal. The other fetus could not be diagnosed because a recombination between the class I HLA and the 21-OHD loci had occurred in this family.

Prenatal diagnosis of 21-hydroxylase deficiency by the complementary deoxyribonucleic acid probe for cytochrome P-450C-21OH

The availability of the complementary deoxyribonucleic acid probe for cytochrome P-450C-21OH and the identification of specific gene defects in some families with congenital adrenal hyperplasia have made prenatal diagnosis feasible. Deoxyribonucleic acid samples from amniocytes of a fetus at 16 weeks' gestation, one previously affected son, and their parents were digested with the restriction enzymes Tagl or EcoRI and hybridized to the cytochrome P-450C-21OH complementary deoxyribonucleic acid probe. The previously affected son and the fetus both lacked the Tagl 3.7 kb band. At the time of delivery, the second child had a cord blood 17α-hydroxyprogesterone level of 8000 ng/dl. The absence of the 3.7 kb Tagl fragment in affected members of this family made possible the use of deoxyribonucleic acid analysis for prenatal diagnosis.

First trimester prenatal diagnosis of 21-hydroxylase deficiency by linkage analysis to HLA-DNA probes and by 17-hydroxyprogesterone determination.

The close genetic linkage between the gene for congenital adrenal hyperplasia due to 21-hydroxylase (21-OH) deficiency and HLA genes allowed us to use the polymorphism of this system as a marker of the disease. HLA genotyping can be performed by using restriction enzyme fragments hybridized with specific probes instead of serologic methods. In seven pregnancies at risk for 21-OH deficiency, a first trimester prenatal diagnosis has been performed by determining the fetal genotype by linkage analysis of DNA from chorionic villi using HLA class I and class II probes. In four of these pregnancies, determination of 17-OH progesterone in first trimester amniotic fluid afforded a complementary approach to the diagnosis.

Pitfalls of prenatal diagnosis of 21-hydroxylase deficiency (21-OH def) congenital adrenal hyperplasia (CAH)

Hormonal measurements and HLA genotyping of amniotic fluid at midgestation correctly predicted the postnatal dx of CAH in 26 of 29 fetuses at risk for CAH. Of these 26, 6 were predicted to have classical 21-OH def based on elevated amniotic fluid 17-hydroxyprogesterone (17-P) and Δ4-androstenedione (A). These 6 fetuses and their index cases were ultimately proven to have salt-wasting classical 21-OH def. Of 3 HLA typed, genotype was identical to the index case. Normal amniotic fluid 17-P and A in the remaining 20 predicted fetuses unaffected with classical CAH, and these patients have been clinically asymptomatic to date or biochemically proven not to be affected with classical or nonclassical CAH. Of the 20 fetuses, 6 were HLA typed and predicted to be homozygous unaffected or heterozygous. However, in 3 of the 29 fetuses, prenatal diagnosis was incorrect. In one, the fetus was predicted to have CAH based on HLA identity to the index case, but amniotic fluid 17-P and A were normal and the fetus was normal. The index case of this family did not have CAH but was a normal child. Thus, amniotic fluid hormone levels accurately predicted a normal fetus while HLA typing was not relevant in prenatal dx because the index case was unaffected. The second fetus was predicted to be a carrier on the basis of HLA typing and normal amniotic fluid 17-P and A. However, during infancy the female infant was shown to have nonclassical CAH and be HLA identical to the index case. The index case in this family, presumed to have classical CAH, was later diagnosed to have nonclassical CAH. In the third case, the fetus was predicted to be a heterozygote by HLA genotyping and to be unaffected by hormonal measurement. Postnatally, at age 2 7/12 yrs, the male child was shown to have non-salt-losing classical CAH and to be HLA identical to the index case who also has non-salt-losinq CAH. These data demonstrate that in salt-losing classical CAH, prenatal dx of the homozygous affected fetus is reliable by the measurement of amniotic fluid 17-P and A. In nonclassical or non-salt-losing classical CAH, amniotic fluid 17-P and A are in the normal range, thus prenatal dx is not possible by hormonal evaluation. Both hormonal and HLA studies of fetal amniotic fluid, as well as correct dx of the specific form of 21-OH def and HLA studies of the index case, are essential in diagnosing the specific form of 21-OH def CAH in the fetus.

Congenital adrenal hyperplasia

Congenital adrenal hyperplasia (CAH) is a family of disorders produced by enzymatic deficiencies of adrenal steroidogenesis. Oversecretion of ACTH secondary to impairment of cortisol synthesis stimulates excessive synthesis of the precursors of the enzyme defect and accumulation of the products of those adrenal hormones whose synthesis is unimpaired by the enzyme deficiency. The most frequent enzyme deficiency is that of 21-hydroxylation. Deficiencies of the enzymes 11β-hydroxylase, 3β-hydroxysteroid dehydrogenase (3β-HSD), 17α-hydroxylase, and cholesterol desmolase has also been reported. Ambiguous genitalia presenting at birth is the most important clinical feature of CAH due to 21-hydroxylase and 11β-hydroxylase deficiencies in genetic females as a result of excessive secretion of adrenal androgens prenatally. In 3β-HSD, 17β-hydroxylase, and cholesterol desmotase deficiency, androgen synthesis is impaired and thus cirilization of the genitalia in genetic males is incomplete. Salt-wasting is associated with those forms of CAH with deficient mineralocorticoid production; in other forms, hypertension may result from accumulation of steroid precursors with salt-retaining properties. Defects of 18-hydroxylase, and 18- dehydrogenase cause a defect in atdosterone synthesis, while 17-hydroxysteroid reductase, and 17–20 lyase deficiencies cause androgen deficiency without interrupting cortisol synthesis. With respect to regulation and secretion, a recent concept is that the adrenal zona fasciculata and zona glomerulosa function as two separate glands: the fasciculata is primarily regulated by ACTII and the glomerulosa by the reninangiotensin system. It has been documented that in CAH due to the 11β-hydroxylase deficiency there is a defect in 11- and 18-hydroxylation in the fasciculata while the enzymatic activity of these enzymes in the glomerulosa is normal. The concept that the fasciculata and glomerulosa function as two separate glands has been applied to CAH due to 21-hydroxylase deficiency. A new hypothesis to explain the presence or absence of salt-wasting signs in CAH due to 21-hydroxylase deficiency is as follows: i) in both simple virilizers and saltwasters there is a fasciculata defect of 21-hydroxylation in both the 17-hydroxy and 17-desoxy pathways, and ii) in saltwasters there is a 21-hydroxylase defect in the glomerulosa as well, while in simple virilizers the glomerulosa is spared this defect. The ability of simple virilizers, but not saltwasters, to respond to renin-angiolensin stimulation with an increase in aldosterone secretion supports this hypothesis. Close genetic linkage between HLA and 21-hydroxylase deficiency has been recently demonstrated, and the 21-hydroxylase deficiency gene has been mapped in close proximity to the HLA-B genetic locus. HLA genotyping, in addition to hormonal testing, is useful for the detection of heterozygote carriers for 21-hydroxylase deficiency and prenatal diagnosis in families with an affected index case. Two non-classical, FILA-linked forms of 21-hydroxylase deficiency are described: the late onset form and the asymptomatic “cryptic” form. Hormonal standards are presented for genotyping 21-hydroxylase deficiency, demonstrating a spectrum in the degree of enzymatic deficiency ranging from a severe deficiency in the classical form to a milder deficiency in the late onset and cryptic disorders, to a still milder deficiency detectable only with ACTII stimulation in heterozygotes for all three forms. It is also proposed that the cryptic disorder represents a genetic compound of the classical and mild 21-hydroxylase deficiency alleles. Studies of genetic linkage disequilibrium suggest that some late onset patients may also be genetic compounds, possessing both a mild and a classical 21-hydroxylase deficiency allele. A microfiller paper method for measuring 17-OHP for the neonatal diagnosis of 21-hydroxylase deficiency is described. The results of a pilot newborn screening program for CAH using the 17-OHP microfilter paper method demonstrates that screening for this disorder is feasible. Further 21-hydroxylase deficiency occurs with sufficient frequency to warrant screening. Measurement of 17-OHP and Δ4 in amniotic fluid make possible the prenatal diagnosis of 21-hydroxylase deficiency. The fundamental aim of therapy in CAH is to provide replacement of the deficient hormones. Glucocorticoid administration both replaces the deficient cortisol and suppresses ACTH production, resulting in a decrease in adrenal androgens which produce virilization and ultimate short stature. Mineralocortocoid supplement is required in the salt-wasting disorders. Mineralocorlicoid administration is also recommended for patients with elevated plasma renin activity but no evident symptoms of salt-wasting, to improve the control of androgen oversecretion. Recognition of this disorder is important in the sex assignment of newborns with ambiguous genitalia.