Prenatal sonography can predict degree of placental invasion.

OBJECTIVE: To evaluate whether the maximum degree of placental invasion (placenta accreta, increta or percreta) can be predicted with ultrasound imaging, using criteria developed in our department. METHODS: This was a retrospective study of all 232 patients at risk for placental invasion who were part of a routine screening program for placental invasion from January 2001 to January 2011. The whole placenta was scanned in a systematic manner using both gray-scale ultrasound and color-flow mapping. Sonographic findings were compared with the clinical outcome during and after delivery and the histomorphological examination of the placenta. RESULTS: Placental invasion was suspected by ultrasound in 40 (17.2%) patients and was clinically/histopathologically confirmed in a total of 35 (15.1%) patients. The sensitivity, specificity and positive and negative predictive values of ultrasound for placental invasion were 91.4% (95% CI, 77.6-97.0%), 95.9% (95% CI, 92.2-97.9%), 80.0% (95% CI, 65.2-89.5%) and 98.4% (95% CI, 95.5-99.5%), respectively. No case of placenta increta (n = 7) or percreta (n = 17) was diagnosed as showing normal placentation or placenta accreta on ultrasound, giving an overall accuracy for the differentiation between normal placentation/placenta accreta and placenta increta/percreta of 100%. CONCLUSION: Our data suggest that prediction of the degree of placental invasion is possible using prenatal ultrasound, with high overall accuracy.

Δ133p53 is an independent prognostic marker in p53 mutant advanced serous ovarian cancer.

BACKGROUND: We aimed to evaluate the clinical relevance of p53 and p73 isoforms that modulate the function of p53. METHODS: This prospective multicentre study included 154 patients with stage III and IV serous ovarian cancer. A functional yeast-based assay and subsequent sequencing were performed to analyse the p53 mutational status. Expression of p53 and p73 isoforms was determined using RT-qPCR. RESULTS: Δ133p53 expression constituted an independent prognostic marker for recurrence-free (hazard ratio=0.571, P=0.016, 95% CI: 0.362-0.899) and overall survival (hazard ratio=0.365, P=0.004, 95% CI: 0.182-0.731) in patients with p53 mutant ovarian cancer (n=121). High Δ40p53 expression was associated with favourable tumour grading (P=0.037) and improved recurrence-free survival (33.4 vs 19.6 months, P=0.029), but not overall survival (43.1 vs 33.6 months, P=0.139), in patients with p53 wild-type cancer (n=33). Neither the p53 mutational status nor p73 isoform expression possessed prognostic significance in the examined ovarian cancer cases. CONCLUSION: Δ133p53 expression was associated with prognosis in the vast majority of ovarian cancer cases, that is, patients with p53 mutant advanced serous carcinomas. Thus, our findings underline the importance of considering the complex p53 regulatory network.

Demographics and anthropometrics impact benefits of health intervention: data from the Reduce Obesity and Diabetes Project.

OBJECTIVE: To determine the efficacy of a 4-month school-based health, nutrition and exercise intervention on body fatness and examine possible effects of demographic and anthropometric covariates. METHODS: Height, weight, waist circumference and body composition were measured in a diverse population of 644 NYC middle school students (mean ± SD age 12.7 ± 0.9 years; 46% male; 38% Hispanic, 17% East Asian, 15% South Asian, 13.5% African American, 8.5% Caucasian, 8% other) during the fall and spring semesters. Year 1 participants (n = 322) were controls. Experimental participants (year 2, n = 469) received a 12-session classroom-based health and nutrition educational programme with an optional exercise intervention. RESULTS: Groups were demographically and anthropometrically similar. The intervention resulted in significant reductions in indices of adiposity (ΔBMI z-scores [-0.035 ± 0.014; p = 0.01], Δ% body fat [-0.5 ± 0.2; p < 0.0001] and Δwaist circumference [-0.73 ± 0.30 cm; p < 0.0001]). Intervention effects were greater (p = 0.01) in men (ΔBMI z-score = -0.052 ± 0.015) versus women (0.022 ± 0.018), participants who were obese (ΔBMI z-score -0.083 ± 0.022 kg m-2) versus lean (-0.0097 ± 0.020 kg m-2) and South Asians (Δ% body fat -1.03 ± 0.35) versus total (-0.49 ± 0.20%) participants (p = 0.005). CONCLUSION: A 4-month school-based health intervention was effective in decreasing measures of adiposity in middle school students, particularly in men, participants who were obese and South Asians.

Genetics of adrenal steroid 21-hydroxylase deficiency.

Impairment of 21-hydroxylation is the most common enzymatic deficiency resulting in the syndrome of CAH, which may present either in the classical form in infants or in the nonclassical form in older individuals. Variable signs and symptoms of androgen excess are common to both types of the disorder, which are transmitted as autosomal recessive traits linked to HLA. Virilization begins in the second month of gestational life in classical 21-OHD, but postnatally in the nonclassical form. Salt wasting is a feature of the disease in a large number of classical patients; in the simple virilizing form aldosterone biosynthesis, a function of the adrenal zona glomerulosa, is intact. Additionally, no patient with nonclassical 21-OHD has been found to have salt wasting. Levels of precursor hormones are less markedly elevated in nonclassical 21-OHD, reflecting a less severe enzyme deficiency; coordinates of basal and stimulated 17-OHP are plotted on a nomogram to ascertain diagnostic category within a family. Confirmatory evidence of heterozygosity within the family of an affected proband is found by performing HLA typing. Specific linkage disequilibria exist for the classical and nonclassical forms of 21-OHD. Frequency of the classical disease is 1/5,000-1/15,000 in Caucasians, whereas the nonclassical disease is found in approximately 1/100 individuals in the Caucasian population, placing the latter disorder among the most common autosomal recessive disorders in man. A deletion of the active 21-hydroxylase gene has been detected in some classical patients; further investigations are in progress to elucidate the molecular genetics of this disease.

Congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

More than 90% of cases of congenital adrenal hyperplasia (CAH, the inherited inability to synthesize cortisol) are caused by 21-hydroxylase deficiency. Females with severe, classic 21-hydroxylase deficiency are exposed to excess androgens prenatally and are born with virilized external genitalia. Most patients cannot synthesize sufficient aldosterone to maintain sodium balance and may develop potentially fatal "salt wasting" crises if not treated. The disease is caused by mutations in the CYP21 gene encoding the steroid 21-hydroxylase enzyme. More than 90% of these mutations result from intergenic recombinations between CYP21 and the closely linked CYP21P pseudogene. Approximately 20% are gene deletions due to unequal crossing over during meiosis, whereas the remainder are gene conversions--transfers to CYP21 of deleterious mutations normally present in CYP21P. The degree to which each mutation compromises enzymatic activity is strongly correlated with the clinical severity of the disease in patients carrying it. Prenatal diagnosis by direct mutation detection permits prenatal treatment of affected females to minimize genital virilization. Neonatal screening by hormonal methods identifies affected children before salt wasting crises develop, reducing mortality from this condition. Glucocorticoid and mineralocorticoid replacement are the mainstays of treatment, but more rational dosing and additional therapies are being developed.

Disease expression and molecular genotype in congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

Genotyping for 10 mutations in the CYP21 gene was performed in 88 families with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Southern blot analysis was used to detect CYP21 deletions or large gene conversions, and allele-specific hybridizations were performed with DNA amplified by the polymerase chain reaction to detect smaller mutations. Mutations were detected on 95% of chromosomes examined. The most common mutations were an A----G change in the second intron affecting pre-mRNA splicing (26%), large deletions (21%), Ile-172----Asn (16%), and Val-281----Leu (11%). Patients were classified into three mutation groups based on degree of predicted enzymatic compromise. Mutation groups were correlated with clinical diagnosis and specific measures of in vivo 21-hydroxylase activity, such as 17-hydroxyprogesterone, aldosterone, and sodium balance. Mutation group A (no enzymatic activity) consisted principally of salt-wasting (severely affected) patients, group B (2% activity) of simple virilizing patients, and group C (10-20% activity) of nonclassic (mildly affected) patients, but each group contained patients with phenotypes either more or less severe than predicted. These data suggest that most but not all of the phenotypic variability in 21-hydroxylase deficiency results from allelic variation in CYP21. Accurate prenatal diagnosis should be possible in most cases using the described strategy.

Reproductive outcome of women with 21-hydroxylase-deficient nonclassic adrenal hyperplasia.

CONTEXT: Because many women with 21-hydroxylase (21-OH)-deficient nonclassic adrenal hyperplasia (NCAH) carry at least one allele affected by a severe mutation of CYP21, they are at risk for giving birth to infants with classic adrenal hyperplasia (CAH). OBJECTIVE: Our objective was to determine the frequency of CAH and NCAH infants born to mothers with 21-OH-deficient NCAH. DESIGN AND SETTING: We conducted an international multicenter retrospective/prospective study. PATIENTS AND METHODS: The outcome of 203 pregnancies among 101 women with 21-OH-deficient NCAH was reviewed. The diagnosis of 21-OH-deficient NCAH was established by a basal or post-ACTH-stimulation 17-hydroxyprogesterone level of more than 10 ng/ml (30.3 nmol/liter). When possible, genotype analyses were performed to confirm CAH or NCAH in the offspring. RESULTS: Of the 203 pregnancies, 138 (68%) occurred before the mother's diagnosis of NCAH and 65 (32%) after the diagnosis. Spontaneous miscarriages occurred in 35 of 138 pregnancies (25.4%) before the maternal diagnosis of NCAH, and in only four of 65 pregnancies (6.2%) after the diagnosis (P < 0.002). Four (2.5%; 95% confidence interval, 0.7-6.2%) of the 162 live births were diagnosed with CAH. To date, 24 (14.8%; 95% confidence interval, 9.0-20.6%) children, 13 girls and 11 boys, have been diagnosed with NCAH. The distribution of NCAH children and their mothers varied significantly by ethnicity (P < 0.0001, for both). CONCLUSIONS: The risk of a mother with 21-OH-deficient NCAH for giving birth to a child affected with CAH is 2.5%; at least 14.8% of children born to these mothers have NCAH.

Mapping of DNA amplifications at 15 chromosomal localizations in 1875 breast tumors: definition of phenotypic groups.

DNA amplification is frequent in breast cancer and has been associated with specific clinicopathological parameters and/or worsened course of the disease. In the present work, we were interested in further defining the association linking the occurrence of DNA amplification to the emergence of specific breast tumor phenotype. To this aim, we studied by Southern blotting a total of 1875 breast tumor DNAs with 26 probes mapping at 15 distinct chromosomal localizations. Of the 26 loci tested, 11 loci showed elevated levels of amplification, 9 loci showed occasional and/or low level of DNA copy number increase, and 6 loci showed very rare or no variation. This allowed us to define six amplified domains mapping at 8p12, 8q24, 11q13, 12q13, 17q12, and 20q13.2, respectively. Over 60% of the tumors analyzed presented at least one amplification at one of these localizations. Amplifications often covered large regions of DNA and bore complex patterns involving coamplification of several colocalized markers. Statistical analysis revealed correlations associating DNA amplification with breast tumor phenotype, as well as sets of preferential coamplifications. Based on these correlations, we defined three subsets of breast cancer according to their patterns of DNA amplification. The first subset (group A) was organized around the amplifications at 11q13 and/or 8p12 and was predominantly composed of estrogen receptor-positive tumors and presented a large proportion of lobular cancers. The second subset (group B) was organized around the amplifications of ERBB2 and/or MYC. These tumors were mostly estrogen receptor-negative and of the ductal invasive type. The third subset (group C) corresponded to tumors in which no amplification was detected in the present screen. Tumors in this group were largely diploid and of low histopathological grading.

Effect of vitamin E supplementation on platelet thromboxane A2 production in type I diabetic patients. Double-blind crossover trial.

Vitamin E deficiency is associated with increased platelet aggregation, which can be normalized through vitamin E supplementation. In diabetes, increased platelet thromboxane A2 (TXA2) production is correlated with decreased platelet vitamin E content. We therefore investigated the effect of 400 mg DL-alpha-tocopherol acetate daily for 4 wk on ADP- and collagen-induced platelet aggregation and platelet TXA2 production in 22 type I (insulin-dependent) diabetic patients without macroangiopathy and with no or only minimal microangiopathy by a double-blind placebo-controlled crossover study. Platelet aggregation was induced in platelet-rich plasma by two or three different concentrations of ADP and collagen. TXA2 was measured by the stable spontaneous breakdown product thromboxane B2 by a specific radioimmunoassay. Whereas metabolic control remained unchanged during the study period, platelet TXA2 production was significantly (P less than .05 and P less than .01) reduced at each ADP concentration and at two of three collagen concentrations. Because increased TXA2 production of diabetic platelets is thought to play an important pathogenetic role in diabetic angiopathy, we conclude that vitamin E treatment could be beneficial with respect to platelet-vessel-wall interaction and thus might be promising for the prevention of diabetic angiopathy.

CD44v6 as a prognostic factor in cervical carcinoma FIGO stage IB.

The aim of our study was to clarify whether CD44v6 evaluation can serve as a universally applicable prognostic factor in patients with FIGO stage IB cervical carcinoma. A retrospective study was performed on 178 FIGO stage IB (142 IB N0, 36 IB N1) radically operated cervical carcinoma patients. The expression of CD44v6 was investigated by immunohistochemistry (IHC). The prognostic significance of established prognostic factors and CD44v6 expression was analyzed by univariate and multivariate analyses. To test the reproducibility and to account for interobserver variability, all specimens were evaluated independently at two institutions. Two different IHC scoring systems, several cut-off levels for CD44v6 positivity and several statistical methods for IHC results evaluation were used. In a univariate analysis, the most significant prognostic factor for overall survival (OS) was lymph node status (p<0.001) followed by tumor volume, LVSI, GOG score (p<0.01) and a deep stromal invasion (p = 0.06). We found a strong correlation between CD44v6 expression and squamous cell carcinoma (SCC) (SCC vs. adenocarcinoma - p<0.001) and between CD44v6 expression and deep stromal invasion, LVSI and GOG score (p<0.05). The CD44v6 expression was not a statistically significant prognostic factor for OS in a univariate analysis (p=0.39 Vienna; p=0.54 Freiburg). In a multivariate analysis, the most significant prognostic factor for OS was lymph node status (p =0.002), followed by tumor diameter and LVSI (p<0.05). CD44v6 expression was not a statistically significant prognostic factor for OS or disease-free interval (DFI) independent of the scoring method used. In conclusion, we demonstrated that CD44v6 expression is associated with LVSI, deep stromal invasion and SCC, but has no prognostic influence on OS and DFI in a population of 178 women with FIGO stage IB cervical carcinoma.

Messenger RNA determination of estrogen receptor, progesterone receptor, pS2, and plasminogen activator inhibitor-1 by competitive reverse transcription-polymerase chain reaction in human breast cancer.

Estrogen receptor (ER), progesterone receptor (PR), the estrogen-inducible protein pS2, and plasminogen activator inhibitor-1 (PAI-1) are important prognostic factors in primary breast cancer. The protein concentrations of these factors in breast tumors have been well documented. However, few data about the mRNA expression of ER, PR, pS2, and PAI-1 in breast cancer are available, which is mostly due to the limitations of conventional techniques for mRNA analysis. We have described a competitive reverse transcription-PCR system for the simultaneous quantification of ER, PR, pS2, and PAI-1 mRNA in tumor samples. Here, we evaluated 100 tumor biopsies from breast cancer patients for the mRNA expression of ER, PR, pS2, and PAI-1. The results were analyzed for correlations with protein status and with clinical data. Significant correlations between mRNA expression levels and protein concentrations of all tested markers were found. In only a few cases was there an obvious discordance between the measurable amounts of mRNA and protein, especially for ER and PR. In addition, ER, PR, and pS2 mRNA levels correlated significantly with each other. No correlation between PAI-1 mRNA amount and the expression of the other markers was found. With respect to clinical data, ER and PR mRNA levels were found to be inversely correlated to tumor size and histological grade but not to the lymph node status. pS2 and PAI-1 mRNA expression were not correlated with tumor size, grade, or lymph node involvement. In conclusion, competitive reverse transcription-PCR may be used as an alternative for the study of prognostic factors in human breast cancer and other malignancies. However, before mRNA expression is measured for diagnostics, a presumed concordance of mRNA and protein expression must be evaluated very carefully for every gene.

A mutation (Pro-30 to Leu) in CYP21 represents a potential nonclassic steroid 21-hydroxylase deficiency allele.

The mild nonclassic form of steroid 21-hydroxylase deficiency is one of the most common autosomal recessive disorders in humans, occurring in almost 1% of caucasians and about 3% of Ashkenazi Jews. Many patients with this disorder carry a Val-281----Leu missense mutation in the CYP21 gene. This and most other mutations causing 21-hydroxylase deficiency are normally present in the CYP21P pseudogene and have presumably been transferred to CYP21 by gene conversion. To identify other potential nonclassic alleles, we used recombinant vaccinia virus to express two mutant enzymes carrying the mutations Pro-30----Leu (normally present in CYP21P) and Ser-268----Thr (considered a normal polymorphism of CYP21). Whereas the activity of the protein carrying the Ser----Thr mutation was indeed indistinguishable from the wild type, the enzyme with the Pro----Leu substitution had 60% of wild-type activity for 17-hydroxyprogesterone and about 30% of normal activity for progesterone when assayed in intact cells. When kinetic analysis of the latter mutant enzyme was performed in cellular lysates, the first order rate constants (maximum velocity/dissociation constant) for both substrates were reduced 10- to 20-fold compared with those for the wild-type enzyme. Pro-30 is conserved in many microsomal P450 enzymes and may be important for proper orientation of the enzyme with respect to the aminoterminal transmembrane segment. The Pro----Leu mutation was present in 5 of 18 patients with nonclassic 21-hydroxylase deficiency, suggesting that this mutation indeed acts as a nonclassic deficiency allele.

Genotype and hormonal phenotype in nonclassical 21-hydroxylase deficiency.

In nonclassical steroid 21-hydroxylase deficiency, the genotype may be represented as a homozygous mild (nonclassical) form of the 21-hydroxylase defect or as a compound heterozygote, with one severe (classical) and one mild (nonclassical) 21-hydroxylase deficiency allele. We examined hormone levels in patients with nonclassical 21-hydroxylase deficiency in whom pedigree analysis and/or HLA linkage disequilibrium allowed unequivocal identification of the respective haplotypes as either classical or nonclassical. The results indicated that compound heterozygotes (21-OH defsevere/21-OH defmild) have an ACTH-stimulated 17-hydroxyprogesterone (17-OHP) response significantly greater than that of mild homozygotes (21-OH defmild/21-OH defmild): at 60 min, 8,131 +/- 4,205 (+/-SD) (n = 17) vs. 4,468 +/- 2,123 ng/dl (n = 31) for the respective groups (P less than or equal to 0.01); at 360 min, 11,067 +/- 5,582 (n = 17) vs. 5746 +/- 1565 (n = 8, P less than or equal to 0.01). Since serum cortisol levels were the same in both groups, the ratio of 17-OHP to cortisol was higher in the former group. Sixty minute ACTH-stimulated serum delta 4-androstenedione levels also were significantly higher in compound heterozygotes than in mild homozygotes. Serum dehydroepiandrosterone and its sulfate were not significantly different between the two groups. Notably, compound heterozygotes were no more likely to have signs of androgen excess than were homozygotes for the mild gene defect. Stimulated levels of serum 17-OHP, 17-OHP/cortisol, delta 4-androstenedione and dehydroepiandrosterone and its sulfate did not differ significantly between heterozygotes for the classical (21-OH defsevere/21-OHnormal) and nonclassical (21-OH defmild/21-OHnormal) 21-hydroxylase deficiency alleles. Thus, the presence of a single normal 21-hydroxylase allele is sufficient to obscure the difference between a severe and a mild 21-hydroxylase deficiency allele on the opposite haplotype. We conclude that the compound heterozygous patients as a group have a significantly higher response of 21-hydroxylase precursors to ACTH stimulation than do patients with the homozygous mild 21-hydroxylase deficiency state.

Insulin insensitivity in adrenal hyperplasia due to nonclassical steroid 21-hydroxylase deficiency.

To determine whether hyperandrogenism caused by an inborn error of adrenal steroidogenesis could produce insulin resistance, we examined insulin sensitivity in females with 21-hydroxylase deficiency. Minimal modelling was used to analyze the results of tolbutamide-modified, frequently sampled, iv glucose tolerance testing. Insulin sensitivity [Si; (min-1) (microU/mL)-1] was plotted against body mass index (BMI; defined as kilograms per m2). Six patients with nonclassical 21-hydroxylase deficiency (mean age, 27 yr; mean BMI, 23.2) underwent testing. None of these patients was in active puberty, nor was any patient being treated with glucocorticoids at the time of the study. Twelve eumenorrheic nonhyperandrogenic young adult female control subjects (mean age, 27 yr; mean BMI, 22.4) were also tested. The basal 17-hydroxyprogesterone concentration, but not the total serum testosterone level, was significantly different in the two groups (mean +/- SEM, 11,987 +/- 2,761 vs. 4,059 +/- 802 pmol/L; P < 0.05). As a group the patients' Si values were significantly lower than those of the controls (mean +/- SEM, 4.1 +/- 0.6 vs. 9.7 +/- 1.2; P < 0.05). There was no correlation between Si and basal serum 17-hydroxyprogesterone, testosterone, delta 4-androstenedione, or dehydroepiandrosterone. We conclude that chronic hypersecretion of androgen precursors due to an inborn error of metabolism can induce a reduction in insulin sensitivity.

Ovarian hyperthecosis in the setting of portal hypertension.

Hepatocellular dysfunction and perturbed portal hemodynamics alter steroid metabolism. Men with liver disease have gynecomastia, although women similarly affected rarely show virilization. We report a 10-yr-old girl with portal hypertension and shunting associated with precocious puberty and ovarian hyperandrogenism. This was one of premature twin girls; neither had clitoromegaly or genital ambiguity. In one child, neonatal respiratory problems led to umbilical vein catheterization with subsequent development of portal hypertension. Pubic hair was first noted at age 6 yr, breasts at 7 yr, and severe acne and clitoromegaly at 10 yr. Baseline sex hormones were elevated: androstenedione (A), 413 ng/dL; testosterone (T), 226 ng/dL; and estradiol (E2), 160 pg/mL. Liver transaminases were within the normal range, however, the coagulation profile was mildly abnormal. Cosyntropin adrenal stimulation revealed no steroidogenic defect. Dexamethasone suppression reduced A and T slightly. LH-releasing hormone stimulation produced a pubertal rise in LH and FSH. Pelvic sonography showed a large right ovary with numerous follicles. Surgical exploration revealed symmetrically enlarged ovaries with dense capsules. Histology of ovarian wedge resections showed hyperthecosis; immunohistochemistry showed stromal cells expressing steroidogenic enzymes and proteins. One month postoperatively, A and T were unchanged from baseline, whereas E2 decreased to 56 pg/mL. A single dose of depot leuprolide acetate significantly reduced T. Subsequent treatment with oral contraceptives reduced T to 50 ng/dL, and cyclical menses occurred. We conclude that precocious puberty and ovarian hyperthecosis were induced in this young girl by elevated circulating levels of sex hormones, a consequence of portasystemic shunting and impaired hepatic steroid metabolism.

Excess mineralocorticoid receptor activity in patients with dexamethasone-suppressible hyperaldosteronism is under adrenocorticotropin control.

Total mineralocorticoid activity in human serum was assessed by the rat renal slice receptor assay (RRA). RRA values were compared to RIA-derived aldosterone (aldo) equivalents. Our data demonstrate that in normal subjects, mineralocorticoid receptor-binding steroids can be almost totally accounted for by immunoreactive deoxycorticosterone, corticosterone, cortisol, and aldo (RRA, 4.73 +/- 1.34 ng/ml aldo; RIA, 3.91 +/- 1.52 ng/ml aldo equivalents), while in 8 patients with dexamethasone-suppressible hyperaldosteronism (DSH), RRA values were greater than RIA values in the basal state (RRA, 7.57 +/- 0.75; RIA, 3.24 +/- 0.34; P less than 0.01). DSH patients had a RRA to RIA ratio after ACTH stimulation similar to the ratio in these patients in the basal state (basal, 2.34; ACTH-stimulated, 2.04). During dexamethasone treatment, RRA values fell markedly (1.82 +/- 0.26). Thus, total mineralocorticoid activity, as measured by RRA in DSH patients, was greater than RIA-measurable deoxycorticosterone, corticosterone, cortisol, and aldo, indicating the presence of an unidentified steroid which is dexamethasone suppressible and ACTH stimulable.

Genotyping of CYP21, linked chromosome 6p markers, and a sex-specific gene in neonatal screening for congenital adrenal hyperplasia.

We investigated the feasibility and diagnostic utility of genotyping 9 CYP21 mutations, linked chromosome 6p markers, and a dimorphic X-Y marker from neonatal screening samples. Blood-impregnated filter papers (Guthrie cards) from 603 randomly chosen New Zealand neonates were genotyped blind to 17-hydroxyprogesterone (17-OHP) levels. Another 50 samples from Swiss and North American infants with correlative hormonal data were also genotyped. DNA was extracted, and gene-specific PCR was performed. CYP21 PCR products were subjected to ligase detection reaction, simultaneously analyzing 9 CYP21 mutations; PCR products of other genes were subjected to direct gel analysis. CYP21 genotyping indicated a heterozygote rate of 2.8% for classic mutations (excluding CYP21 deletions), and 2.0% for nonclassic mutations in New Zealanders. Ten full-term affected neonates showed a wide range of 17-OHP levels (15-1400 nmol/L). Sick or preterm infants or infants screened on the first day of life with high 17-OHP proved genetically unaffected. Genetic linkage disequilibrium was found between two CYP21 mutations and chromosome 6p markers. Guthrie cards can be used to accurately genotype CYP21 and other relevant markers, potentially enhancing the specificity and sensitivity of congenital adrenal hyperplasia screening. CYP21 heterozygote frequency for classic mutations is higher than expected based on genotype compared with that predicted by hormonal newborn screening.

Study of a kindred with classic congenital adrenal hyperplasia: diagnostic challenge due to phenotypic variance.

We sought to determine the concordance of the phenotype and genotype in a kindred with classic congenital adrenal hyperplasia due to 21-hydroxylase deficiency. The variation in phenotypic expression within this family underscores the difficulty of establishing the diagnosis in the absence of newborn screening, even with a heightened index of suspicion. Steroidogenic profiles were obtained for the three affected siblings. The available clinical history of the two affected aunts was retrieved. Genotyping was performed on several members of the kindred. Detailed sequencing of the entire CYP21 gene of two clinically dissimilar subjects in this family was undertaken to explore the possibility of other mutations or polymorphisms. PCR with ligase detection reaction analysis of CYP21 revealed that the affected family members III-2, III-3, III-4, II-3, and II-4, all were compound heterozygotes carrying the intron 2 point mutation known to interfere with splicing (nucleotide 656 A to G) and the exon 4 point mutation causing a nonconservative substitution of asparagine for isoleucine at codon 172 (I172N). Detailed sequencing of the gene was performed for the two most phenotypically dissimilar subjects. A single silent polymorphism was found in the third nucleotide for codon 248 in patient II-4, but not in patient III-4, and no additional mutations were found. Classic congenital adrenal hyperplasia remains a difficult diagnosis to make in the absence of newborn screening because of the variability of phenotypic expression. Likewise, the variable degree of genital ambiguity in affected females in this family serves to question universal advocacy of prenatal steroid treatment in pregnancies at risk for congenital adrenal hyperplasia. Extensive molecular exploration did not provide an explanation of the phenotypic heterogeneity and supports the possibility of influences other than the CYP21 gene for the observed divergence.

Prevalence of nonclassical steroid 21-hydroxylase deficiency based on a morning salivary 17-hydroxyprogesterone screening test: a small sample study.

Early morning salivary 17 alpha-hydroxyprogesterone (17-OHP) determination differentiates patients with non-classical 21-hydroxylase deficiency (NC21OHD) from those who are not affected. Using this test, we have conducted a trial screening study for NC21OHD and have compared the study results with previously reported figures for the frequency of this disorder. Testing was performed on 258 subjects recruited from among the medical students and employees of the New York Hospital-Cornell Medical Center. In 2 of the 249 admissible subjects, the 0700-0900 h salivary 17-OHP level was within the range for NC21OHD patients (0.72-6.7 nmol/L; n = 8). These 2 individuals were subsequently confirmed to be affected by ACTH testing. Of the subjects with morning salivary 17-OHP levels below the cut-off point of 0.72 nmol/L, 29 were recalled for ACTH testing and were confirmed to be unaffected. Prevalence of NC21OHD in the test population was determined according to ethnic group. Our study gives a prevalence by screening of 1.14% among caucasians, which agrees with values of 0.81% and 1.06% obtained by different analytical methods. Further, both affected subjects were Ashkenazi Jews, and the prevalence of 3.23% among study members from this group concurs with increased rates of 3.64% and 4.97% already reported. On the basis of a small population sample, screening so far confirms the claim that NC21OHD is the most common autosomal recessive human disorder. Using values from ACTH-proven unaffected subjects (n = 47) and NC21OHD patients (n = 10), we establish preliminary normative data for morning salivary 17-OHP levels of 0.172 nmol/L for unaffected subjects (95% confidence interval, 0.05-0.54 nmol/L) and 1.76 nmol/L for NC21OHD-affected subjects (95% confidence interval, 0.42-7.32 nmol/L).

First trimester prenatal treatment and molecular genetic diagnosis of congenital adrenal hyperplasia (21-hydroxylase deficiency).

Prenatal treatment of pregnancies at risk for congenital adrenal hyperplasia due to 21-hydroxylase deficiency was carried out in conjunction with chorionic villus sampling (CVS) in the first trimester for analysis of restriction fragment length polymorphisms. Fourteen families of a total of 49 families at risk for this disease elected to undergo both prenatal treatment and diagnosis via CVS. Dexamethasone administration to the pregnant woman was initiated at a mean gestational age of 7 weeks (range, 4-10 weeks) before testing to determine whether the fetus was affected with 21-hydroxylase deficiency, and CVS was performed at a gestational age of 8-10 weeks. Two affected female fetuses were identified by molecular genetic techniques among this group; neonatal physical examination demonstrated amelioration of the degree of genital ambiguity compared with both nonprenatally treated older sisters with 21-hydroxylase deficiency. The duration of unnecessary prenatal dexamethasone treatment for unaffected or male fetuses was substantially reduced in the CVS group compared with that in a cohort of 8 prenatally treated pregnancies in which amniocentesis was performed in the early second trimester. There were no major morbidities observed in the treated pregnancies. Postnatal confirmation of CVS diagnosis was obtained in all cases in which DNA from an affected sibling was available for comparative analysis with the DNA from chorionic villus tissue. We conclude based on these data that the benefit/risk ratio is favorable for prenatal administration of dexamethasone in pregnancies at risk for 21-hydroxylase deficiency. Treatment should be initiated during the first trimester in conjunction with diagnosis by CVS/molecular genetic techniques. Long term postnatal surveillance is recommended for all offspring of dexamethasone-treated pregnancies.