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Patients with congenital adrenal hyperplasia (CAH) often suffer from long-term complications secondary to chronic glucocorticoid therapy and suboptimal treatment regimens.The aim of the study was to describe clinical characteristics of a large cohort of pediatric and adult CAH patients.We conducted a cross-sectional study of 244 CAH patients [183 classic, 61 nonclassic (NC)] included in a Natural History Study at the National Institutes of Health.Outcome variables of interest were height sd score, obesity, hypertensive blood pressure (BP), insulin resistance, metabolic syndrome, bone mineral density, hirsutism (females), and testicular adrenal rest (TART).The majority had elevated or suppressed androgens, with varied treatment regimens. Mean adult height SD score was -1.0 ± 1.1 for classic vs. -0.4 ± 0.9 for NC patients (P = 0.015). Obesity was present in approximately one third of patients, across phenotypes. Elevated BP was more common in classic than NC patients (P ≤ 0.01); pediatric hypertensive BP was associated with suppressed plasma renin activity (P = 0.001). Insulin resistance was common in classic children (27%) and adults (38% classic, 20% NC); 18% of adults had metabolic syndrome. The majority (61%) had low vitamin D; 37% of adults had low bone mineral density. Hirsutism was common (32% classic; 59% NC women). TART was found in classic males (33% boys; 44% men).Poor hormonal control and adverse outcomes are common in CAH, necessitating new treatments. Routine monitoring of classic children should include measuring BP and plasma renin activity. Osteoporosis prophylaxis and TART screening should begin during childhood. A longitudinal study is under way.

BACKGROUND Chimeric CYP21A1P/CYP21A2 genes, caused by homologous recombination between CYP21A2 (cytochrome P450, family 21, subfamily A, polypeptide 2) and its highly homologous pseudogene CYP21A1P (cytochrome P450, family 21, subfamily A, polypeptide 1 pseudogene), are common in patients with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD). A comprehensive junction site analysis of chimeric CYP21A1P/CYP21A2 genes is needed for optimizing genetic analysis strategy and determining clinical relevance. METHODS We conducted a comprehensive genetic analysis of chimeric CYP21A1P/CYP21A2 genes in a cohort of 202 unrelated 21-OHD patients. Targeted CYP21A2 mutation analysis was performed, and genotyping of chimeric CYP21A1P/CYP21A2 genes was cross-confirmed with Southern blot, RFLP, and multiplex ligation-dependent probe amplification analyses. Junction sites of chimera genes were determined by sequencing the long-PCR products amplified with primers CYP779f and Tena32F. An updated bioinformatics survey of Chi-like sequences was also performed. RESULTS Of 100 probands with a chimeric allele, 96 had a chimera associated with the severe classic salt-wasting form of CAH, and the remaining 4 carried an uncommon attenuated chimera with junction sites upstream of In2G (c.293–13A/C>G), which is associated with a milder phenotype. In addition to 6 of 7 reported chimeras, we identified a novel classic chimera (CH-8) and a novel attenuated chimera (CH-9). Attenuated chimeras explained prior genotype–phenotype discrepancies in 3 of the patients. Sequencing the CYP779f/Tena32F amplicons accurately differentiated between classic and attenuated chimeras. The bioinformatics survey revealed enrichment of Chi-like sequences within or in the vicinity of intron 2. CONCLUSIONS Junction site analysis can explain some genotype–phenotype discrepancies. Sequencing the well-established CYP779f/Tena32F amplicons is an unequivocal strategy for detecting attenuated chimeric CYP21A1P/CYP21A2 genes, which are clinically relevant.

ObjectiveTo comprehensively phenotype parents identified with nonclassic congenital adrenal hyperplasia (NCCAH) by family genetic studies, termed here as cryptic NCCAH and to define the incidence of cryptic NCCAH in the parents of a large cohort of patients with 21-hydroxylase deficiency.DesignGenotyping was performed on 249 parents of 145 unrelated congenital adrenal hyperplasia (CAH) patients. Parents with two CYP21A2 mutations underwent extensive evaluation.ResultsOf the 249 parents, ten (4%; seven females and three males) were identified as having cryptic NCCAH. The majority was of ethnicities previously reported to have a higher incidence of NCCAH. Cosyntropin stimulation performed in eight parents provided biochemical confirmation (17-hydroxyprogesterone range 56–364 nmol/l) and cortisol response was ≤500 nmol/l in three parents (38%). Of the seven women (27–54 years) with cryptic NCCAH, four had prior infertility, two reported irregular menses, two had treatment for hirsutism, one had androgenic alopecia. Men were asymptomatic. All cryptic NCCAH parents reported normal puberty and had normal height. Adrenal hypertrophy and a small adrenal myelolipoma were observed in two parents; testicular adrenal rest tissue was not found.ConclusionsParents diagnosed with NCCAH by genetic testing are mostly asymptomatic. Temporary female infertility and suboptimal cortisol response were commonly observed. Ongoing glucocorticoid therapy is not indicated in adults with CAH identified by family genotype studies unless symptomatic, but glucocorticoid stress coverage should be considered in select cases. Parents of a child with CAH have a 1:25 risk of having NCCAH; if the mother of a child with CAH has infertility, evaluation for NCCAH is indicated.

The mammalian growth plate undergoes programmed senescence during juvenile life, causing skeletal growth to slow with age. We previously found that hypothyroidism in rats slowed both growth plate chondrocyte proliferation and growth plate senescence, suggesting that senescence is not dependent on age per se but rather on chondrocyte proliferation. However, one alternative explanation is that the observed slowing of growth plate senescence is a specific consequence of hypothyroidism. We reasoned that, if delayed senescence is a general consequence of growth inhibition, rather than a specific result of hypothyroidism, then senescence would also be slowed by other growth-inhibiting conditions. In this study, we therefore used tryptophan deficiency to temporarily inhibit growth in newborn rats for 4 weeks. We then allowed the animals to recover and studied the effects on growth plate senescence. We found that structural, functional, and molecular markers of growth plate senescence were delayed by prior tryptophan deficiency, indicating that the developmental program of senescence had occurred more slowly during the period of growth inhibition. Taken together with previous studies in hypothyroid rats, our findings support the hypothesis that delayed senescence is a general consequence of growth inhibition and hence that growth plate senescence is not simply a function of time per se but rather depends on growth.

Background: Genetic analysis is commonly performed in patients with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. Study Objective: The objective of the study was to describe comprehensive CYP21A2 mutation analysis in a large cohort of CAH patients. Methods: Targeted CYP21A2 mutation analysis was performed in 213 patients and 232 parents from 182 unrelated families. Complete exons of CYP21A2 were sequenced in patients in whom positive mutations were not identified by targeted mutation analysis. Copy number variation and deletions were determined using Southern blot analysis and PCR methods. Genotype was correlated with phenotype. Results: In our heterogeneous U.S. cohort, targeted CYP21A2 mutation analysis did not identify mutations on one allele in 19 probands (10.4%). Sequencing identified six novel mutations (p.Gln262fs, IVS8+1G>A, IVS9-1G>A, p.R408H, p.Gly424fs, p.R426P) and nine previously reported rare mutations. The majority of patients (79%) were compound heterozygotes and 69% of nonclassic (NC) patients were compound heterozygous for a classic and a NC mutation. Duplicated CYP21A2 haplotypes, de novo mutations and uniparental disomy were present in 2.7% of probands and 1.9 and 0.9% of patients from informative families, respectively. Genotype accurately predicted phenotype in 90.5, 85.1, and 97.8% of patients with salt-wasting, simple virilizing, and NC mutations, respectively. Conclusions: Extensive genetic analysis beyond targeted CYP21A2 mutational detection is often required to accurately determine genotype in patients with CAH due to the high frequency of complex genetic variation.

Human phosphodiesterase (PDE) type 8B (PDE8B) is located at 5q14.1 and is known as the PDE with the highest affinity to cAMP. We recently described a family with bilateral micronodular adrenocortical disease that was apparently caused by an inactivating PDE8B mutation (H305P). As a result of a genome-wide study, a strong association between six polymorphic variants in the PDE8B promoter and serum levels of the thyroid-stimulating hormone (TSH) has been recently reported. Despite an extended analysis of the regions surrounding 5q14.1, no other potential genetic variants that could be responsible for the associated TSH levels were found.In this study, we genotyped by polymerase chain reaction the described six polymorphic variants in the PDE8B promoter in the family with micronodular adrenocortical disease and inactivating PDE8B mutation and analyzed their correlation with individual TSH values in the family members.We observed complete segregation between the reported association and individual TSH values in the family we studied. Haplotype analysis showed that the haplotype associated with the high TSH levels is different from the one that segregated with H305P, suggesting that the mutation most probably has arisen on an allele independent of the high TSH-associated allele.The proposed mechanism by which PDE8B may influence TSH levels is through control of cAMP signaling. Our analysis revealed separate segregation of an inactivating PDE8B allele from the high-TSH-allele and showed low TSH levels in persons who carry an inactivating PDE8B allele. These data suggest that, indeed, PDE8B may be involved in regulation of TSH levels.

Mammalian somatic growth is rapid in early postnatal life but then slows and eventually ceases in multiple tissues. We hypothesized that there exists a postnatal gene expression program that is common to multiple tissues and is responsible for this coordinate growth deceleration. Consistent with this hypothesis, microarray analysis identified more than 1600 genes that were regulated with age (1 vs. 4 wk) coordinately in kidney, lung, and heart of male mice, including many genes that regulate proliferation. As examples, we focused on three growth-promoting genes, Igf2, Mest, and Peg3, that were markedly down-regulated with age. In situ hybridization revealed that expression occurred in organ-specific parenchymal cells and suggested that the decreasing expression with age was due primarily to decreased expression per cell rather than a decreased number of expressing cells. The declining expression of these genes was slowed during hypothyroidism and growth inhibition (induced by propylthiouracil at 0–5 wk of age) in male rats, suggesting that the normal decline in expression is driven by growth rather than by age per se. We conclude that there exists an extensive genetic program occurring during postnatal life. Many of the involved genes are regulated coordinately in multiple organs, including many genes that regulate cell proliferation. At least some of these are themselves apparently regulated by growth, suggesting that, in the embryo, a gene expression pattern is established that allows for rapid somatic growth of multiple tissues, but then, during postnatal life, this growth leads to negative-feedback changes in gene expression that in turn slow and eventually halt somatic growth, thus imposing a fundamental limit on adult body size.

In mammals, the rate of somatic growth is rapid in early postnatal life but then slows with age, approaching zero as the animal approaches adult body size. To investigate the underlying changes in cell-cycle kinetics, [methyl-3H]thymidine and 5’-bromo-2’deoxyuridine were used to double-label proliferating cells in 1-, 2-, and 3-week-old mice for four weeks. Proliferation of renal tubular epithelial cells and hepatocytes decreased with age. The average cell-cycle time did not increase in liver and increased only 1.7 fold in kidney. The fraction of cells in S-phase that will divide again declined approximately 10 fold with age. Concurrently, average cell area increased approximately 2 fold. The findings suggest that somatic growth deceleration primarily results not from an increase in cell-cycle time but from a decrease in growth fraction (fraction of cells that continue to proliferate). During the deceleration phase, cells appear to reach a proliferative limit and undergo their final cell divisions, staggered over time. Concomitantly, cells enlarge to a greater volume, perhaps because they are relieved of the size constraint imposed by cell division. In conclusion, a decline in growth fraction with age causes somatic growth deceleration and thus sets a fundamental limit on adult body size.

In mammals, somatic growth is rapid in early postnatal life but decelerates with age and eventually halts, thus determining the adult body size of the species. This growth deceleration, which reflects declining proliferation, occurs simultaneously in multiple organs yet appears not to be coordinated by a systemic mechanism. We, therefore, hypothesized that growth deceleration results from a growth-limiting genetic program that is common to multiple tissues. Here, we identified a set of 11 imprinted genes that show down-regulation of mRNA expression with age in multiple organs. For these genes, Igf2, H19, Plagl1, Mest, Peg3, Dlk1, Gtl2, Grb10, Ndn, Cdkn1c, and SLC38a4, the declines show a temporal pattern similar to the decline in growth rate. All 11 genes have been implicated in the control of cell proliferation or somatic growth. Thus, our findings suggest that the declining expression of these genes contributes to coordinate growth deceleration in multiple tissues. We next hypothesized that the coordinate decline in expression of these imprinted genes is caused by altered methylation and consequent silencing of the expressed allele. Contrary to this hypothesis, the methylation status of the promoter regions of Mest, Peg3, and Plagl1 did not change with age. Our findings suggest that a set of growth-regulating imprinted genes is expressed at high levels in multiple tissues in early postnatal life, contributing to rapid somatic growth, but that these genes are subsequently downregulated in multiple tissues simultaneously, contributing to coordinate growth deceleration and cessation, thus imposing a fundamental limit on adult body size.