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.     

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.     

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.     

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.     

Long-term outcome of patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency

AIMS: Conflicting results exist regarding bone mineral density (BMD), metabolism and reproductive function of adult patients with congenital adrenal hyperplasia (CAH). We evaluated the long-term outcome and the impact of chronic glucocorticoid replacement in these patients. METHODS: Physical characteristics, serum hormone concentrations, BMD and metabolism were studied in 45 consecutive CAH adult patients. RESULTS: Among the 36 women, only 14 (39%) had regular menses. Among the 27 women with classical CAH, the mean number of surgical reconstructions of virilized genitalia was 2.1 +/- 0.2. Twenty of them (74%) were sexually active. Three men presented with testicular adrenal rest tumors. Twenty-five patients (55%) had decreased BMD at the femoral neck and/or at the lumbar spine. BMI was correlated with the BMD T-score at the femoral neck (p < 0.001) and at the lumbar spine (p < 0.01). Hydrocortisone dose was negatively correlated with the BMD T-score at the femoral neck (p = 0.04). Subjects with osteopenia had a significantly lower BMI and received higher hydrocortisone dose than those with normal BMD. Overweight was found in 21 patients (47%). There was a significantly positive correlation between HOMA and BMI (p < 0.001), and between HOMA and 17-OHP levels (p = 0.016). CONCLUSIONS: Adult patients with CAH treated with long-term glucocorticoids are at risk for decreased BMD, increased BMI, and disturbed reproductive function.     

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.     

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.     

Inherited deficiency of delta-aminolevulinic acid dehydratase.

Delta-aminolevulinic acid dehydratase (ALA-D) is the second enzyme in the porphyrin-heme pathway and converts delta-aminolevulinc acid (ALA) to porphobilinogen (PBG). A family is reported with an inherited deficiency of red cell ALA-D activity occurring over three generations in an autosomal dominant pattern. Intial experiments support the hypothesis that the mutation in this family may affect a regulatory gene, but enzyme purification and further study are required. Although no clinical manifestations of deficient ALA-D activity have been found in affected persons, families such as this may be at increased risk for the serious consequences of lead poisoning, which produces marked inhibition of ALA-D activity.     

Inherited erythrocyte phosphofructokinase deficiency: Molecular mechanism

Summary Erythrocyte PFK activity 50–60% that of normal controls was found in a mother and her son, without muscular or hematological symptoms.The PFK activity of the mother's muscle was normal in fresh preparations and partially unstable to storage at 4°C. Electrophoresis of muscle PFK revealed two bands, one normal and one abnormal with an anodic mobility greater than normal. Both patients were characterized as heterozygotes for an unstable muscle PFK. Unstable M'subunits disappeared in erythrocytes which are old cells devoid of protein synthesis. Consequently an increased E/M subunit ratio leads to a distribution of the five isozymes different from that of normal erythrocytes. In these patients, we observed a loss of the M₄ enzyme together with an increase in the E₄ isozyme. The kinetic and immunologic data were compatible with these modifications. Isoelectric focusing of hemolysates from the two patients revealed an acidification of the main activity band, suggesting that an increase in E₄ isozyme resulted in a change of the total electric charge.Unité de recherche d'enzymologie des cellules sanguines (INSERM U160 & ERA 573 du CNRS) Hôpital Beaujon 92118 Clichy Cedex, France     

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.     

Inherited glycosylphosphatidyl inositol deficiency: A treatable CDG

Tethering to cell membrane through attachment to the complex glycolipid anchor glycosylphosphatidyl inositol (GPI) is a mode of protein expression highly conserved in eukaryotes. The evolutionary purpose of such an elaborate way of expressing proteins is not clear and neither is the functional role of GPI itself. GPI-anchored proteins (GPI-AP) serve a variety of functions that include adhesion, receptors, signal transduction and complement activation. GPI biosynthesis, a process that is accomplished in at least 9 steps and involves several proteins, some with enzymatic activity, would be expected to be a fertile ground for development of inherited, autosomal recessive disorders. However, until recently, paroxysmal nocturnal haemoglobinuria, a rare haematological disorder caused by somatic mutations in the X-linked PIGA gene, was the only genetic disorder affecting GPI biosynthesis. Here we review the clinical spectrum, biochemical defect and genetic pathogenesis of inherited GPI deficiency, the first described form of inherited, autosomal recessive disorder of GPI biosynthesis and outline the molecular basis of targeted therapy for this condition.     

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.     

Mineralocorticoids in congenital adrenal hyperplasia

While hypertension is observed in only two of the three major subtypes of congenital adrenal hyperplasia (CAH), 11β- and 17-hydroxylase deficiencies, deoxycorticosterone ( (DOC) production is increased in all. The elevated zona fasciculata (ZF) DOC produces mineralocorticoid hypertension with suppressed renin and reduced potassium concentrations. The DOC levels in 21-hydroxylase deficiency are in part produced by renin stimulation of the Zona glomerulosa (ZG) along with aldosterone. Assessment of the mineralocorticoid hormones of the ZF and ZF (17-deoxy steroids) provides additional unique characteristics of each subtype. Dissociation of DOC from cortisol is not unique to CAH. This dissociation is seen in other disorders and contrived conditions. There is a strong suggestion of a non-ACTH regulator of 17-deoxy steroids (DOC) that may contribute significantly to DOC production in general and effect DOC levels in CAH.     

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.     

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.     

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.