Congenital adrenal hyperplasia: focus on the molecular basis of 21-hydroxylase deficiency

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder caused by defects in one of several steroidogenic enzymes involved in the synthesis of cortisol from cholesterol in the adrenal glands. More than 90% of cases are caused by 21-hydroxylase deficiency, and the severity of the resulting clinical symptoms varies according to the level of 21-hydroxylase activity. 21-Hydroxylase deficiency is usually caused by mutations in the CYP21A2 gene, which is located on the RCCX module, a chromosomal region highly prone to genetic recombination events that can result in a wide variety of complex rearrangements, such as gene duplications, gross deletions and gene conversions of variable extensions. Molecular genotyping of CYP21A2 and the RCCX module has proved useful for a more accurate diagnosis of the disease, and prenatal diagnosis. This article summarises the clinical features of 21-hydroxylase deficiency, explains current understanding of the disease at the molecular level, and highlights recent developments, particularly in diagnosis.     

Congenital adrenal hyperplasia: update on prenatal diagnosis and treatment

The diagnostic term congenital adrenal hyperplasia (CAH) applies to a family of inherited disorders of steroidogenesis caused by an abnormality in one of the five enzymatic steps necessary in the conversion of cholesterol to cortisol. The enzyme defects are translated as autosomal recessive traits, with the enzyme deficient in more than 90% of CAH cases being 21-hydroxylase. In the classical forms of CAH (simple virilizing and salt wasting), owing to 21-hydroxylase deficiency (21-OHD), androgen excess causes external genital ambiguity in newborn females and progressive postnatal virilization in males and females. Non-classical 21-OHD (NC21OHD) refers to the condition in which partial deficiencies of 21-hydroxylation produce less extreme hyperandrogenemia and milder symptoms. Females do not demonstrate genital ambiguity at birth.

The gene for adrenal 21-hydroxylase, CYP21, is located on chromosome 6p in the area of HLA genes. Specific mutations may be correlated with a given degree of enzymatic compromise and the clinical form of 21-OHD. NC21OHD patients are predicted to have mild mutations on both alleles or one severe and one mild mutation of the 21-OH locus (compound heterozygote). In most cases the mutation groups represent one diagnosis (e.g., Del/Del with SW CAH), however we have found several non-correlations of genotype to phenotype. Non-classical and classical patients were found within the same mutation group. Phenotypic variability within each mutation group has important implications for prenatal diagnosis and treatment.

Prenatal treatment of 21-OHD with dexamethasone has been utilized for a decade. An algorithm has been developed for prenatal diagnosis and treatment, which, when followed closely, has been safe for both the mother and the fetus, and has been effective in preventing ambiguous genitalia in the affected female newborn. This is an instance of an inborn metabolic error successfully treated prenatally.

Since 1986, prenatal diagnosis and treatment of congenital adrenal hyperplasia due to 21-hydroxylase deficiency (21-OHD) has been carried out in 403 pregnancies in The New York Hospital–Cornell Medical Center. In 280, diagnoses were made by amniocentesis, while 123 were diagnosed using chorionic villus sampling. Of the 403 pregnancies evaluated, 84 babies were affected with classical 21-OHD. Of these, 52 were females, 36 of whom were treated prenatally with dexamethasone. Dexamethasone administered at or before 10 weeks of gestation (23 affected female fetuses) was effective in reducing virilization. Thirteen cases had affected female sibs (Prader stages 1–4); 6 of these fetuses were born with entirely normal female genitalia, while 6 were significantly less virilized (Prader stages 1–2) than their sibs, and one was Prader stage 3. Eight newborns had male sibs; 4 were born with normal genitalia, 3 were Prader stages 1–2, and 3 were born Prader stages 3–4. No significant or enduring side effects were noted in either the mothers or the fetuses, indicating that dexamethasone treatment is safe. Prenatally treated newborns did not differ in weight, length, or head circumference from untreated, unaffected newborns.

Based on our experience, proper prenatal diagnosis and treatment of 21-OHD is effective in significantly reducing or eliminating virilization in the newborn female. This spares the affected female the consequences of genital ambiguity of genital surgery, sex misassignment, and gender confusion.     

Congenital adrenal hyperplasia: lessons from a multinational study

A study group of paediatric endocrinologists was established in Austria, Czech Republic, Hungary, Slovenia and Slovakia in order to investigate various aspects in children with congenital adrenal hyperplasia (CAH). Five hundred and ninety-eight patients with CAH who were diagnosed between 1969 and 1998 were included in order to analyze the following questions. Epidemiological data: There were significantly fewer males (43%) than females (57%), and the percentage of males did not increase during the observation period. Salt wasters (SW) totalled 64.7%, whereas 35.3% had simple virilizing (SV) CAH. Diagnosis was established significantly later in boys than in girls (median of 26 vs. 13 days for SW, p < 0.0001; 1,817 vs. 1,010 days for SV, p < 0.03). Mortality in the general population was significantly lower than in CAH siblings (1.8% vs. 7.0%, p < 0.0001) or in SW children (2.2% vs. 11.3%, p < 0.0001). According to our calculation with the present clinical diagnostic criteria in Central Europe, from 40 expected CAH patients/year, 2-2.5 SW, and one female and four male SV patients will not be diagnosed. Auxological data: Growth data from 341 patients were analyzed retrospectively. Percentiles were constructed in a longitudinal/cross-sectional study and pubertal growth was described in a longitudinal analysis. Growth of SW patients was impaired in early childhood (0-3 years), but followed a normal course until puberty. In contrast, SV children had a normal growth pattern during early childhood, but were above the standard thereafter. The pubertal growth spurt was of normal magnitude in boys and girls, but started too early. Final height was reduced compared with both standard and target heights. There was no correlation between final height and age of starting treatment or the year of birth. Bone age was accelerated in both CAH types, but more so in SV patients. Molecular genetics: Three hundred and fifty-six patients were investigated for 11-14 of the most frequent mutations by direct allele-specific polymerase chain reaction (PCR) and/or PCR followed by sequence-specific oligonucleotide, single strand chain polymorphism and restriction fragment length polymorphism. In the group as a whole, we most frequently found the Intron 2 splice mutation (30.8%) or a deletion/conversion (28.5%). The Intron 2 mutation was most frequent in the Hungarian population, whereas deletions/conversions were found more frequently in Slovenians. The other mutations had a similar distribution to those seen in other populations. Genotype-phenotype correlation confirms previous reports.     

Bioconversion of lignocellulosic biomass: biochemical and molecular perspectives

In view of rising prices of crude oil due to increasing fuel demands, the need for alternative sources of bioenergy is expected to increase sharply in the coming years. Among potential alternative bioenergy resources, lignocellulosics have been identified as the prime source of biofuels and other value-added products. Lignocelluloses as agricultural, industrial and forest residuals account for the majority of the total biomass present in the world. To initiate the production of industrially important products from cellulosic biomass, bioconversion of the cellulosic components into fermentable sugars is necessary. A variety of microorganisms including bacteria and fungi may have the ability to degrade the cellulosic biomass to glucose monomers. Bacterial cellulases exist as discrete multi-enzyme complexes, called cellulosomes that consist of multiple subunits. Cellulolytic enzyme systems from the filamentous fungi, especially Trichoderma reesei, contain two exoglucanases or cellobiohydrolases (CBH1 and CBH2), at least four endoglucanases (EG1, EG2, EG3, EG5), and one β-glucosidase. These enzymes act synergistically to catalyse the hydrolysis of cellulose. Different physical parameters such as pH, temperature, adsorption, chemical factors like nitrogen, phosphorus, presence of phenolic compounds and other inhibitors can critically influence the bioconversion of lignocellulose. The production of cellulases by microbial cells is governed by genetic and biochemical controls including induction, catabolite repression, or end product inhibition. Several efforts have been made to increase the production of cellulases through strain improvement by mutagenesis. Various physical and chemical methods have been used to develop bacterial and fungal strains producing higher amounts of cellulase, all with limited success. Cellulosic bioconversion is a complex process and requires the synergistic action of the three enzymatic components consisting of endoglucanases, exoglucanases and β-glucosidases. The co-cultivation of microbes in fermentation can increase the quantity of the desirable components of the cellulase complex. An understanding of the molecular mechanism leading to biodegradation of lignocelluloses and the development of the bioprocessing potential of cellulolytic microorganisms might effectively be accomplished with recombinant DNA technology. For instance, cloning and sequencing of the various cellulolytic genes could economize the cellulase production process. Apart from that, metabolic engineering and genomics approaches have great potential for enhancing our understanding of the molecular mechanism of bioconversion of lignocelluloses to value added economically significant products in the future. JIMB 2008: BioEnergy - Special issue.     

Precursor-to-product ratios reflect biochemical phenotype in congenital adrenal hyperplasia

Precursor-to-product ratios in steroid hormone metabolism may accurately reflect enzymatic activity and production of metabolites relative to their disappearance. The purpose of this study was to explore the use of direct precursor-to-product steroid ratios to discriminate between infants with congenital adrenal hyperplasia (CAH) due to 21-α-hydroxylase deficiency and infants with no disorder, thus characterizing the biochemical phenotype in CAH. Deidentified dried blood spot samples from confirmed CAH cases identified by newborn screen (CAH-positive, N = 8) and from cases with no disorder (CAH-negative, N = 10) were obtained from the California State Newborn Screening Program. Samples (~6.25 mm circular spots) underwent methanol and water extraction (9:1 ratio). Deuterated steroids served as isotope internal standards. 17-α-hydroxyprogesterone (17-OHP), 11-deoxycortisol (S), androstenedione (A4) and cortisol (F) concentrations were determined by liquid chromatography–tandem mass spectrometry (LC–MS/MS), and the 17-OHP/S, 17-OHP/A4, and S/F ratios were calculated. The mean 17-OHP and A4 concentrations in samples from CAH cases were significantly increased when compared to cases with no disorder (p = 0.003 for both). 17-OHP/S and 17-OHP/A4 ratios were also significantly elevated in CAH cases (p = 0.007 and p < 0.001, respectively). In contrast, S and F concentrations and the S/F ratio were similar between the two groups. In CAH, the elevated 17-OHP/S ratio is a biomarker of diminished 21-α-hydroxylase activity, and the elevated 17-OHP/A4 ratio is a biomarker of adrenal androgen excess via increased 17,20-lyase activity. The similar S/F ratio indicates that the rate of production via 11-β-hydroxylase and disappearance of F is maintained in CAH.     

A case with combined rare inborn metabolic disorders: Congenital adrenal hyperplasia and ornithine transcarbamylase deficiency

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is a common autosomal recessive disorder. Ornithine transcarbamylase (OTC) deficiency is the most common urea cycle disorder and demonstrates X-linked inheritance. In female OTC deficiency, phenotypes are variable according to X-inactivation patterns. These disorders develop separately, and their co-morbidity is extremely rare. We report one girl with CAH showing recurrent hyperammonemia and hepatitis after 2 years-of-age due to additional OTC deficiency.     

Combined adrenal myelolipoma and medullary hyperplasia

OBJECTIVE: A patient is reported with hypertension due to combined medullary adrenal hyperplasia and myelolipoma. METHODS: A 52-year-old woman with long-standing hypertension was evaluated for an incidentally discovered large tumor of the left adrenal. Left adrenalectomy was performed for a presumptive clinical diagnosis of pheochromocytoma. RESULTS: Histopathologic examination revealed a mixed tumor consisting of a large myelolipoma with infiltrating foci of adrenal medulla. CONCLUSIONS: A patient is described with hypertension, myelolipoma and adrenal medullary hyperplasia; following adrenalectomy, however, blood pressure and biochemical abnormalities normalized.     

Molecular pathogenesis of lipoid adrenal hyperplasia and adrenal hypoplasia congenita

Congenital lipoid adrenal hyperplasia (lipoid CAH) is the most severe form of CAH in which the synthesis of all gonadal and adrenal cortical steroids is markedly impaired. Lipoid CAH may be caused by the defect in either the steroidogenic acute regulatory (StAR) protein or the P450scc. More than 34 different mutations in StAR gene have been identified. Clinically, most of the patients manifest adrenal insufficiency from 1 day to 2 months of age, but some patient show delayed onset of adrenal insufficiency. Affected 46, XY subjects do not show pubertal development, whereas affected 46, XX subjects undergo spontaneous feminization, breast development and cyclical vaginal bleeding at the usual age of puberty.

X-linked adrenal hypoplasia congenital (AHC) is a rare congenital adrenal disorder characterized by severe adrenal insufficiency and hypogonadotropic hypogonadism. More than 80 different several intragenic mutations of DAX-1 have been identified. The failure of pubertal development may be caused by either abnormal hypothalamic or pituitary regulation of gonadotropin secretion. In addition, although the testicular steroidogenesis is largely intact, the functional maturity of Sertoli cells and also spermatogenesis are impaired. The type of mutation does not predict clinical phenotype. Thus, unified mechanism how DAX-1 gene defect gives rise to adrenal insufficiency, hypothalamic/pituitary hypogonadism and impaired spermatogenesis remains established.     

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.     

Plant-aphid interactions: molecular and ecological perspectives

Many aphids are major agricultural pests because of their unparalleled reproductive capacity and their ability to manipulate host plant physiology. Aphid population growth and its impact on plant fitness are strongly influenced by interactions with other organisms, including plant pathogens, endophytes, aphid endosymbionts, predators, parasitoids, ants, and other herbivores. Numerous molecular and genomic resources have recently been developed to identify sources of aphid resistance in plants, as well as potentially novel targets for control in aphids. Moreover, the same model systems that are used to explore direct molecular interactions between plants and aphids can be utilized to study the ecological context in which they occur.     

Theoretical molecular biology: prospectives and perspectives

I briefly discuss some aspects of theoretical molecular biology. Specifically, I include the issues of searches for homologies via string matchings, for patterns of specific nucleotide groupings and of sequence-structure relationship. The various approaches developed in order to achieve this end are described, attempting to convey some of the excitement in this quickly growing field.     

Congenital analbuminaemia: Molecular defects and biochemical and clinical aspects

Background

DNA and mRNA sequencing of the coding regions of the human albumin gene (ALB) and of its intron/exon junctions has revealed twenty-one different molecular defects causing congenital analbuminaemia (CAA).

Scope of review

To describe the mutations in molecular terms and to present the current knowledge about the most important biochemical and clinical effects of CAA.

Major conclusions

CAA is rare, but its frequency seems to be significantly higher in restricted and minimally admixed populations. The condition affects especially the lipid metabolism but apart from a possible increased risk for atherosclerotic complications, it is generally associated with mild clinical symptoms in adults. By contrast, several reports indicate that analbuminaemic individuals may be at risk during the perinatal and childhood periods, in which they seem to show increased morbidity and mortality. The twenty-one causative defects include seven nonsense mutations, seven changes affecting splicing, five frame-shift/deletions, one frame-shift/insertion and one mutation in the start codon. These results indicate that the trait is an allelic heterogeneous disorder caused by homozygous (nineteen cases) or compound heterozygous (single case) inheritance of defects. Most mutations are unique, but one, named Kayseri, is responsible for about half of the known cases.

General significance

Study of the defects in the ALB resulting in CAA allows the identification of “hot spot” regions and contributes to understanding the molecular mechanism underlying the trait. Such studies could also give molecular information about different aspects of ALB regulation and shed light on the regulatory mechanisms involved in the synthesis of the protein. This article is part of a Special Issue entitled Serum Albumin.     

Staphylococcal lipases: biochemical and molecular characterization

To date, the nucleotide sequences of nine different lipase genes from six Staphylococcus species, three from S. epidermidis, two from S. aureus, and one each from S. haemolyticus, S. hyicus, S. warneri, and S. xylosus, have been determined. All deduced lipase proteins are similarly organized as pre-pro-proteins, with pre-regions corresponding to a signal peptide of 35 to 38 amino acids, a pro-peptide of 207 to 321 amino acids with an overall hydrophilic character, and a mature peptide comprising 383 to 396 amino acids. The lipases are secreted in the pro-form and are afterwards processed to the mature form by specific proteases. The pro-peptide of the S. hyicus lipase is necessary for efficient translocation and for protection against proteolytic degradation. Despite being very similar in their primary structures the staphylococcal lipases show significant differences in their biochemical and catalytic properties, such as substrate selectivity, pH optimum and interfacial activation. The lipase from S. hyicus is unique among the staphylococcal and bacterial lipases in that it has not only lipase activity, but also a high phospho-lipase activity. All staphylococcal lipases are dependent on Ca(2+), which is thought to have a function in stabilizing the tertiary structure of the lipases. Evidence exists that staphylococcal lipases like other bacterial lipases, possess a lid-like domain that might be involved in the interfacial activation of these enzymes.