Genetic aspects of congenital adrenal hyperplasia due to 21-hydroxylase deficiency

The variability of clinical and biological expression of the 21 OH hydroxylase deficiency is likely to be related to genetical variability. Beside the well known autosomic recessive mode of inheritance the frequencies of the different forms of the disease, especially the classical and late onset form, have been more precisely defined through neonatal screening programs for the classical form which lead to a frequency of about 1 case/20,000 with a calculated gene frequency around 1/140. The linkage with the major histocompatibility complex allows the location of the putative locus of the 21 OH ase on the short arm of the chromosome 6 in the class III of the MHC. This linkage has made possible a better fetal diagnosis even if some pitfalls as recombination must be kept in mind. On the basis of clinical conditions the abnormal genes are likely to be considered as an allelic series with a least two main types of pathological alleles: the "severe" and "moderate". During the last two years, taking advantages of molecular gene biology, the structure of the normal human 21 OH ase gene has been studied. It exists as duplicate genes in close relation with the gene of the fourth component of the complement. A deletion of one of the copy has been demonstrated in the form associated with the BW47 MHC haplotype. It is likely that during the coming years genetical heterogeneity will be demonstrated as it has been for other genetic diseases as thalassemia.     

Cognitive outcome in adult women affected by congenital adrenal hyperplasia due to 21-hydroxylase deficiency

BACKGROUND: Some research suggests that girls with congenital adrenal hyperplasia (CAH), who are exposed to higher than normal levels of prenatal androgens, perform better on spatial tasks, worse on verbal tasks and have a greater incidence of left-handedness than unaffected controls, all of which suggests the development of a more male-typical cognitive pattern. However, research in all three areas has produced inconsistent findings. OBJECTIVES: To determine if prenatal androgen exposure has an organizing effect on female cognitive development and to what extent. METHODS: 24 women, 21-71 years, with either the salt-losing (SL) or simple virilizing (SV) forms of CAH due to 21-hydroxylase deficiency, and 18 controls, 21-73 years, who were unaffected female relatives or women with polycystic ovary syndrome, were assessed with IQ, handedness, executive function, verbal learning and memory, non-verbal learning and memory, perceptual speed, visuospatial processing and visuomotor ability measures. The battery included tests known to elicit sex differences and control measures. RESULTS: The findings did not support the hypothesis that women with CAH develop a more male-typical cognitive pattern. CONCLUSION: This study differs from others in the older age of its participants, grouping by SL/SV diagnosis and assessment of medical treatment and compliance as determined through hormone assays. Our findings provide additional support for the conclusion that, in adult women with CAH, previous prenatal androgen exposure does not enhance spatial abilities, impair verbal abilities nor alter hand preferences in a long-lasting way.     

Genotype of Yupik Eskimos with congenital adrenal hyperplasia due to 21-hydroxylase deficiency

Summary An A-to-G transition in the second intron was the sole mutation detected in four Yupik Eskimo patients with salt-wasting congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency. Allele-specific hybridization should be an efficient means of performing prenatal diagnosis of the disease in this highly inbred population.     

Classical forms of congenital adrenal hyperplasia due to 21-hydroxylase deficiency in adults

During childhood, the main aims of the medical treatment of congenital adrenal hyperplasia (CAH) secondary to 21-hydroxylase are to prevent salt loss and virilization and to attain normal stature and normal puberty. As such, there is a narrow therapeutic window through which the intended results can be achieved. In adulthood, the clinical management has received little attention, but recent studies have shown the relevance of long-term follow-up of these patients. The aims here are to review the multiple clinical, hormonal and metabolic abnormalities that could be found in adult CAH patients as such a decrease in bone mineral density, overweight and disturbed reproductive functions. In women with classic CAH, a low fertility rate is reported, and is probably the consequence of multiple factors including neuroendocrine and hormonal factors, feminizing surgery, and psychological factors. Men with CAH may present hypogonadism either through the effect of adrenal rests or from suppression of gonadotropins resulting in infertility. Therefore a multidisciplinary team with knowledge of CAH should carefully follow up these patients, from childhood through to adulthood, to avoid these complications and to ensure treatment compliance and tight control of the adrenal androgens.     

Procedure for neonatal screening for congenital adrenal hyperplasia due to 21-hydroxylase deficiency

The value of screening of neonates for congenital adrenal hyperplasia is not universally accepted. Procedures for screening are recommended here in order to provide a structure to the testing and ultimately bring together data that will allow the effect of screening to be judged for benefit or dismissed as no better than clinical recognition of the disease state.     

Sharing of MHC haplotypes among apparently unrelated patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency

From the study of HLA, complement, and glyoxalase I alleles in 82 Venezuelan individuals belonging to 19 families of mixed ethnic origin having 20 affected newborns with salt-wasting congenital adrenal hyperplasia due to 21-hydroxylase (21-OH) deficiency, a total of 38 disease haplotypes and 53 nondisease haplotypes were found. Of the pathological haplotypes 47 % were found to share the HLA-B39 or -Bw62 specificities, 55 % of them in combination with the BFS, C2C, C4A4, C4B2 (SC42) complotype. The frequencies of HLA-B39 and -Bw62 among the affected haplotypes were 29 and 18% as compared with 6 and 0 % among the nondisease haplotypes of the same families. Statistical associations (P < 0.01) with salt-wasting adrenal hyperplasia were found with the SC42 complotype and with the combination SC42, HLA-B39. These results are markedly different from those reported in the literature which show an association at the population level among many Caucasoid samples of HLA-Bw47 and the extended haplotype (HLA-Bw47, DR7, FC91, 0) with the salt-wasting form of the disease. Furthermore, four of the unrelated patients reported here were homozygous for all the major histocompatibility complex loci tested, while three others were homozygous for at least two HLA loci. Analysis of the geographical origin of the grandparents indicated clustering of the deficiency carrier HLA haplotypes. This observation, together with the fact that there is an excess of homozygotes among the patients in Venezuela, strongly suggests that salt-wasting 21-OH deficiency congenital adrenal hyperplasia is mostly the result of a founder effect of relatively few independent mutations and, thus, of identity by descent of a few abnormal alleles at the 21-OHB locus in most cases. The mutation marked by HLA-Bw47 was not observed in this population.     

Molecular analysis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency in Hong Kong Chinese patients

BackgroundCongenital adrenal hyperplasia (CAH) caused by 21-hydroxylase deficiency (21OHD) is an autosomal recessive disorder due to mutation in the CYP21A2 gene.ObjectiveTo elucidate the genetic basis of 21-hydroxylase-deficient CAH in Hong Kong Chinese patients.Patients and methodsMutational analysis of the CYP21A2 gene was performed on 35 Hong Kong Chinese patients with 21OHD using direct DNA sequencing and multiplex ligation-dependent probe amplification (MLPA).ResultsThe genetic findings of 21 male and 14 female patients are the following: c.293-13A/C>G (intron 2 splice site; 20 alleles), p.I172N (13), p.R356W (7), p.Q318X (4). A total of 20 mutant alleles contained gross deletion/conversion of all or part of the CYP21A2 gene. A novel mutation, c.1367delA (p.D456fs), was detected in one patient. One patient had only a heterozygous mutation detected. Out of 35 patients, 16 would have been incorrectly genotyped if either DNA sequencing or MLPA alone was used for molecular analysis.ConclusionsThe frequency of various mutations in the studied patients differs from those reported in other Asian populations. Gross deletion/conversion accounts for nearly one-third of the genetic defects. Therefore, laboratories must include methods for detecting point mutations as well as gross deletions/conversions to avoid misinterpretation of genotype. Genotyping has increasingly been proven to be a useful tool for supplementing, if not replacing, hormonal profiling for the diagnosis of 21OHD.     

Increased salt appetite in patients with congenital adrenal hyperplasia 21-hydroxylase deficiency

Salt appetite was investigated in 14 patients with congenital adrenal hyperplasia of the salt-wasting form (SW group), 12 patients with the simple virilized form who are not salt losing, and 18 healthy siblings. Salt appetite was evaluated by questionnaire, preference tests, and dietary analyses. The findings showed that SW who were not therapeutically normalized showed increased salt appetite but no change in sweet preference. Their salt appetite correlated with symptoms of salt wasting, namely, plasma renin activity, plasma K(+), and urine Na(+) and (inversely) with blood pressure. Sensitivity to the taste of NaCl was not altered. Factor analyses of a larger group confirmed the distinction between salt appetite and sweet preference, but intake of dietary Na(+) and sweet carbohydrates and intake of salty and sweet snacks did not reflect distinct salt or sweet preferences. We confirm that putative perinatal dehydration, due to maternal nausea and vomiting during pregnancy, childhood vomiting, and diarrhea with occasional saline infusion, was related to increased salt appetite in adolescence. The findings suggest that salt appetite in humans is determined by interdependent, innate, physiological, and acquired attributes. Salt appetite in SW patients is an adaptive response mediated by the renin-angiotensin system, an innate predisposition to acquire salt preference (in anticipation of both sodium loss and its consequence), and imprinting by perinatal hyponatremic occurrences. Our findings contribute to understanding human salt intake, provide insight into the motivation for salt in patients with congenital adrenal hyperplasia 21-OH deficiency, and may point the way to improvements in therapeutic compliance in these patients.     

Normal bone mineral content in young adults with congenital adrenal hyperplasia due to 21-hydroxylase deficiency

OBJECTIVE: To assess bone mineral content (BMC) and body composition in adolescents and young adults with congenital adrenal hyperplasia. METHODS: BMC, anteroposterior-projected bone area (BA), bone mineral density, and fat mass percentage were determined by dual-energy X-ray absorptiometry in 18 patients aged 18-33 years (8 females and 10 males) with 21-hydroxylase deficiency using a Hologic QDR 1000/W densitometer. RESULTS: BMC and bone mineral density for age were significantly reduced at -1.2 standard deviation scores (SDS; range from -2.8 to +4.1) and -1.1 SDS (range from -3.2 to +2.6), respectively. The BA for height was significantly increased at +1.7 SDS (range from -0.5 to +4.8), and the BMC for BA was normal at 0.3 SDS (range from -2.0 to +3.6). The median final height was significantly reduced at -1.6 SDS (range from -5.6 to +0.3), and the fat mass percentage was significantly increased at +1.5 SDS (range from -1.0 to +5.0). CONCLUSION: Our study population of young adults with 21-hydroxylase deficiency had a short stature and broad bones with a normal BMC and had an increased fat mass percentage.     

Molecular genetics of 21-hydroxylase deficiency in congenital adrenal hyperplasia

21-hydroxylase gene analysis was performed on the genomic DNA from patients with congenital adrenal hyperplasia (CAH), their siblings, their parents as well as from a healthy individual serving as control. After digestion by the Taq I and Bgl II restriction enzymes, DNA was hybridized with specific nucleotidic probes: pC21a for the 21-hydroxylase genes, pAT-A for the C4 component Complement genes, closely linked to the 21-hydroxylase genes on the 6 chromosome. Likewise the pFB3B probe was used for the B factor gene located 80 kilobases upstream the 21-hydroxylase gene. From this molecular analysis on 11 families, we report here 4 investigations showing the most frequent genetic abnormalities we have encountered: gene deletions, gene conversions and point mutations. These data show that the molecular approach is a powerful tool for studying this endocrine disease at the clinical, genetic and fundamental point of view.     

Serum 17-hydroxypregnenolone and 17-hydroxypregnenolone sulfate concentrations in patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency

Serum concentrations of 17-hydroxypregnenolone, 17-hydroxypregnenolone sulfate and 17-hydroxyprogesterone were measured simultaneously in patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency, using a combined radioimmunoassay method. All these precursor steroids were found to be markedly elevated in the sera of untreated patients with a salt-losing form of the disease, whereas, in untreated patients with a simple virilizing form, only the concentration of unconjugated steroids was increased and the 17-hydroxypregnenolone sulfate concentration remained within the normal range. Among the patients with a salt-losing form under maintenance therapy, these steroids were all still significantly increased in those on insufficient control, whereas only 17-hydroxyprogesterone was significantly but slightly increased in those on adequate control. Although the mechanism whereby the serum 17-hydroxypregnenolone sulfate concentration is not increased in the untreated simple virilizers is unknown, both a milder degree of 21-hydroxylase deficiency and a role of 17-hydroxypregnenolone sulfate in adrenal steroid production as a kind of supplier are suggested as possible explanations, especially in the neonatal period and early infancy. Thus, this study showed the serum concentrations of 17-hydroxypregnenolone and its sulfate together with 17-hydroxyprogesterone in patients with 21-hydroxylase deficiency in various conditions.     

Mutational analysis in Lebanese patients with congenital adrenal hyperplasia due to a deficit in 21-hydroxylase

Molecular defects in the gene encoding steroid 21-hydroxylase (CYP21) result in impairment of adrenal steroid synthesis in patients affected with autosomal-recessive congenital adrenal hyperplasias (CAH). In this study, we report on the molecular screening of six point mutations, large deletions, gene conversion events and duplications in 25 unrelated Lebanese families affected by CAH due to steroid 21-hydroxylase. The methods used (PCR-digestion and southern blot) allowed the detection of 96% of the disease chromosomes. In classical forms, the most frequent mutation was the splice site mutation in intron 2 accounting for 39% of the disease alleles. Gene conversion events accounted for 14% of the alleles, but no large deletions were found. In nonclassical forms, the V281L mutation in exon 7 represent 86% of the tested alleles. Genotype-phenotype correlations were as expected: Delta 8nt, Q318X and gene conversion correspond to SW forms, whereas the intron 2 splice site mutation may give either SW or SV forms; the V281L mutation was responsible for nonclassical forms. The spectrum of mutations underlines the genetic diversity of the Lebanese population. No correlation could be drawn out between mutations and some specific religious communities, except for the Delta 8nt mutation, which is present only in the Christian Maronite group. Molecular study of the CYP21 gene might constitute a good support for clinicians, especially in consanguineous families, for whom we could provide genetic counselling.     

Changes of several adrenal delta 4-steroids measured by HPLC-UV spectrometry in neonatal patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency

We have developed an easy and rapid method of reverse-phase high-performance liquid chromatography (HPLC)-UV spectrometry for measuring adrenal delta 4-steroids. Three female neonates with adrenal 21-hydroxylase deficiency (2 salt-losers and 1 simple virilizer), two of whom were recalled by neonatal mass-screening for congenital adrenal hyperplasia (CAH), were diagnosed using this method. Changes of several adrenal steroids were examined in these patients before and after treatment with hydrocortisone. Before treatment, the cortisone and cortisol peaks were very low and those of 17 alpha-hydroxyprogesterone (17-OHP) and 21-deoxycortisol (21-DOF) were high in all 3 patients (17-OHP: 79.9-997 nmol/l, 21-DOF: 83.7-324 nmol/l). The androstenedione peak was also high in 2 of them. A peak produced by 21-deoxycortisone, which is a product of oxidation of 21-DOF at the C-11 position, was also detected in all cases (14.5-297 nmol/l). After treatment, all of these abnormally elevated delta 4-steroids decreased or disappeared. This new method is thought to be valuable for the rapid diagnosis of CAH, and especially for use in neonatal mass-screening for CAH.     

Prenatal diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency by allele-specific hybridization and Southern blot

The feasibility and accuracy of gene-specific molecular genetic diagnosis for congenital adrenal hyperplasia due to 21-hydroxylase deficiency was studied in a group of 24 pregnancies at 25% risk of carrying an affected fetus. Chorionic villus sampling was performed at 9–10 weeks' gestation. Southern analysis and polymerase chain reaction, followed by allele-specific hybridization for a panel of nine known mutations, were performed for each family. Mutations were identified in 95% of chromosomes examined; the molecular diagnosis was accurate in 96% of infants as confirmed by postnatal examination. The most common mutation identified was an A-to-G transition at base 656 in the second intron, the result of an apparent gene conversion. In one family, there had been a de novo mutation in intron 2, which was detected in the proband, but not in the mother or in the fetus. We conclude that first trimester prenatal diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency is feasible and accurate employing CYP21-specific probes.     

A rational,non-radioactive strategy for the molecular diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency

Context

Molecular diagnosis of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21OHD) has not been straightforward.

Objective

To conduct a comprehensive genetic analysis by Multiplex Ligation dependent Probe Amplification (MLPA) and evaluate its reliability for the molecular CAH-21OHD diagnosis.

Patients and methods

We studied 99 patients from 90 families with salt-wasting (SW; n = 32), simple-virilizing (SV; n = 29), and non-classical (NC; n = 29) CAH-21OHD. Molecular analysis was sequentially performed by detecting the most frequent point mutations by allele-specific oligonucleotide polymerase chain reaction (ASO-PCR), large rearrangements by MLPA, and rare mutations by direct sequencing. Parental segregation was evaluated.

Results

ASO-PCR detected microconversions in 164 alleles (91.1%). MLPA identified CYP21A1P large conversions to CYP21A2 in 7 of the remaining 16 (43.7%), 30-kb deletions including the 3′-end of CYP21A1P, C4B, and the 5′-end of CYP21A2 in 3 of the 16 (18.7%), and a complete CYP21A2 deletion in one (6.3%). Five alleles (2.7%) required direct sequencing; three mutations located in the CYP21A2 gene and two derived from CYP21A1P were found. No parental segregation was observed in patients with the c.329_336del and/or the CL6 cluster mutations. These cases were not diagnosed by ASO-PCR, but MLPA detected deletions in the promoter region of the CYP21A2 gene, explaining the genotype/phenotype dissociation.

Conclusion

Using the proposed algorithm, all alleles were elucidated. False-positive results in MLPA occurred when mutations or polymorphisms were located close to the probe-binding regions. These difficulties were overcome by the association of MLPA with ASO-PCR and paternal segregation. Using these approaches, we can successfully use MLPA in a cost-effective laboratory routine for the molecular diagnosis of CAH-21OHD.     

Complement component 4 copy number variation and CYP21A2 genotype associations in patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD) is an autosomal recessive disorder of cortisol biosynthesis caused by CYP21A2 mutations. An increase in gene copy number variation (CNV) exists at the CYP21A2 locus. CNV of C4, a neighboring gene that encodes complement component 4, is associated with autoimmune disease susceptibility. In this study, we performed comprehensive genetic analysis of the RP-C4-CYP21-TNX (RCCX) region in 127 unrelated 21-OHD patients (100 classic, 27 nonclassic). C4 copy number was determined by Southern blot. C4 CNV and serum C4 levels were evaluated in relation to CYP21A2 mutations and relevant phenotypes. We found that the most common CYP21A2 mutation associated with the nonclassic form of CAH, V281L, was associated with high C4 copy number (p?=?7.13?×?10^?16). Large CYP21A2 deletion, a common mutation associated with the classic form of CAH, was associated with low C4 copy number (p?=?1.61?×?10^?14). Monomodular RCCX with a short C4 gene, a risk factor for autoimmune disease, was significantly less frequent in CAH patients compared to population estimates (2.8 vs. 10.6?%; p?=?1.08?×?10^?4). In conclusion, CAH patients have increased C4 CNV, with mutation-specific associations that may be protective for autoimmune disease. The study of CYP21A2 in relation to neighboring genes provides insight into the genetics of CNV hotspots, an important determinant of human health.     

Five patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency (one with associated neuroblastoma) discovered in three generations of one family

BACKGROUND: Most patients with 21-hydroxylase deficiency (21-OHD) are compound heterozygous carriers. Their phenotype usually reflects a less severe allelic mutation, although discordance between the genotype and the phenotype has been observed. CASE REPORT: We present 5 patients with congenital adrenal hyperplasia (CAH) due to 21-OHD belonging to the 3 generations of the same family (grandmother, parents and their 2 children). As each patient carries at least one mild mutation of the CYP21 gene, their genotypes correspond to nonclassical CAH. The propositus is the older brother, who is compound heterozygous with a mild and severe CYP21 mutation (P30L/R356W). In spite of one mild CYP21 mutation, he presented with the clinical picture of a simple virilizing form of 21-OHD and required glucocorticoid replacement therapy from the age of 4. Both probands' parents are compound heterozygous carriers of different CYP21 gene mutations causing various degrees of enzymatic activity impairment, which explains the different genotypes and phenotypes in their offspring. The probands' mother, besides the nonclassical 21-OHD, also had neuroblastoma of the adrenal gland. CONCLUSION: The potential discordance between the genotype and the phenotype in some patients with CAH is emphasized. The existence of a mild CYP21 mutation P30L in a compound heterozygous with CAH might be associated with progressive virilization requiring glucocorticoid therapy from early childhood. The occurrence of neuroblastoma with 21-OHD may support the hypothesis that an impairment in the synthesis and secretion of glucocorticoids may play role in the development and functioning of the adrenal medulla.     

Prenatal diagnosis of 21-hydroxylase deficiency congenital adrenal hyperplasia using the polymerase chain reaction

Summary We present an improved method for the prenatal diagnosis of congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency. The polymerase chain reaction (PCR) was used to analyze DNA from an affected index case, the parents, and a cultured chorionic villus sample, for point mutations in the steroid 21-hydroxylase (CYP21) gene. We can predict that the fetus is an unaffected carrier.     

Growth patterns and final height in congenital adrenal hyperplasia due to classical 21-hydroxylase deficiency. Results of a multicenter study

BACKGROUND: Longitudinal growth and bone age (BA) development are the most important clinical parameters for monitoring adequate glucocorticoid replacement in children with congenital adrenal hyperplasia (CAH). AIM OF THE STUDY: To analyze the growth pattern of patients treated for CAH of the salt wasting (SW) and simple virilizing (SV) clinical forms; to evaluate final height as compared to reference data and individual target height; to evaluate the course of BA development. PATIENTS AND METHODS: A large database of 598 patients with CAH was created in 5 Central European countries and growth data of 341 treated patients with 21-hydroxylase deficiency were analyzed retrospectively. The patients were of Caucasian origin. Centiles were constructed in a cross-sectional manner and an additional longitudinal analysis was performed in order to evaluate the pubertal growth spurt by applying particular statistical methods (Preece-Baines model). RESULTS: The growth of SW CAH patients was impaired in infancy and early childhood (0-3 years of age), but followed normal patterns in childhood until puberty. In contrast, children with SV CAH had normal patterns of growth in infancy and early childhood and were considerably taller than healthy references during childhood. In the longitudinal study, peak height velocity in both boys and girls was normal, but it occurred at an earlier age than in the standard population. The final height of patients with CAH was reduced in comparison to both the reference and the individual target height. No correlations were found between final height and age at the start of the therapy in SV patients or between final height and year of birth. BA was advanced in both types of CAH, but more accelerated in SV patients. CONCLUSION: Characteristic growth patterns for treated SV and SW CAH children were identified, with a normal pubertal growth spurt and reduced final height being observed.