Growth in height and body proportion from birth to adulthood in hereditary hypophosphatemic rickets: a retrospective cohort study.

PURPOSE: Patients with hereditary hypophosphatemic rickets are short and disproportionate and very little information is available on segmental growth, but the body disproportion at adulthood leads us to think that the growth velocity of legs is slower. METHODS: A total of 96 children were included and molecular testing was carried out in 42. Children who reached adult height were classified into two groups according to their compliance to conventional treatment (phosphate supplement and calcitriol). Individual growth records of height and sitting height/height were plotted using Argentine reference data in 96 children and growth curves were estimated by fitting Preece-Baines Model 1 in 19 of the children. RESULTS: Molecular testing revealed sequence deleterious alterations or large deletions in 36/42 patients. During childhood, 76% of children grew below - 1.88 standard deviation score (SDS) and 97% had body disproportion. During adolescence, the mean peak height velocity for the good and poor compliance to treatment groups was 7.8 (0.6) and 5.4 (0.4) cm/year in boys and 7.0 (0.7) and 5.2 (0.8) cm/year in girls, respectively. At adulthood, the median sitting height/height ratio was 2.32 and 6.21 SDS for the good and poor compliance to treatment groups, respectively. The mean pubertal growth spurt of the trunk was -0.8 (1.4) SDS, with a short pubertal growth spurt of - 1.8 (0.4) SDS for limbs in the good compliance group. Median adult height in 13/29 males and 30/67 females was -4.56 and -3.16 SDS, respectively. CONCLUSION: For all patients the growth spurt was slower, secondary to a short growth spurt of limbs, reaching a short adult height with body disproportion that was more prominent in the poor compliance group.

Genetic and clinical spectrum of aromatase deficiency in infancy, childhood and adolescence.

INTRODUCTION: cP450aromatase deficiency provides clues for the understanding of the role of aromatase in prepubertal and pubertal human health and disease. Placental aromatization of androgens protects the female fetus against the virilizing action of fetal androgens. After birth, the dual effect of aromatase deficiency, excessive androgens, and insufficient estrogens is responsible for a variable clinical picture. Nineteen cases of aromatase gene (CYP19) deficiency have been reported. PHENOTYPE: Phenotype is dependent on sex and age. In newborns, aromatase deficiency should be considered in the etiology of 46,XX DSD, after ruling out congenital adrenal hyperplasia. In prepubertal aromatase deficient girls, high levels of ovarian androgens and gonadotropins facilitate the formation of ovarian cysts. Bone mineralization can be affected and bone aging is delayed. In pubertal girls, there is poor sexual development and abnormal virilization. The phenotype may be variable according to enzyme activity level. Insulin sensitivity may be abnormal in both men and women. Finally, aromatase might also play a role in the regulation of testicular cell mass in the newborn testis. CONCLUSION: Adequate interpretation of clinical data should lead to the analysis of the CYP19 gene for diagnostic confirmation and implementation of appropriate management.

Serum IGF-I and IGFBP-3 reference values from a chemiluminescent assay in normal children and adolescents of hispanic and italian origin: presence of sexual dimorphism in IGF-I values.

Serum IGF-I and IGFBP-3 assays are used to monitor rhGH treatment. Some discrepancies in results obtained by means of different assays have been reported. The aim of this study was to establish normal ranges for circulating IGF-I and IGFBP-3 in children and adolescents of Hispanic and Italian origin. Circulating levels of IGF-I and IGFBP-3 were measured in 169 Hispanic and Italian prepubertal children and 66 adolescents of both sexes, using a chemiluminescent assay. Serum levels of IGF-I and IGFBP-3 increased from early childhood into adolescence. After pubertal peaks of IGF-I and IGFBP-3, slight decreases were observed with increasing age. Furthermore, serum IGF-I levels were significantly higher in girls than in boys, suggesting a sexual dimorphism in serum IGF-I values in late prepuberty and early puberty. Differences in IGF-I and IGFBP-3 absolute values between our study and previous studies suggest the need to establish reference ranges for each ethnic group.

Variations in biological and immunological activity of growth hormone during the neonatal period.

BACKGROUND/AIMS: It was postulated that a high growth hormone (GH) bioactivity might explain the rapid growth rate of neonates. The aim of this study is to verify changes in serum GH biological potency (Bio-/Immuno-GH ratio) and their effects on serum growth factors during the first month of life in term and preterm babies. METHODS: Blood samples were collected from 10 small-for-gestational-age preterm (SGAPT), 17 appropriate for gestational age preterm (AGAPT) and 26 AGA term (T) neonates on days 4, 15 and 30 of life to evaluate serum GH values measured by IFMA (IFMA-GH) and bioassay (Bio-GH), serum insulin-like growth factor-I (IGF-I) and IGF-binding protein-3 (IGFBP-3). RESULTS: High serum Bio-GH values on the first few days of life correspond to high IFMA-GH values, suggesting full biological potency of circulating GH. Furthermore, IGF-I/IGFBP-3 molar ratio values in preterm babies were higher than in full-term infants. CONCLUSIONS: These data confirmed the hypothesis that the higher growth velocity in the first month of life of preterm neonates is due to an increased bioavailability of IGF-I. A progressive maturation of the hypothalamic-pituitary-IGF-I axis without any alteration in the GH biological potency seems to underpin the increase of the growth factors early in life.

Progressive increase in non-sex-hormone-binding globulin-bound testosterone from infancy to late prepuberty in boys.

Recent evidence suggests that human albumin-bound testosterone (HSA-bound T), the major constituent of nonsex hormone-binding globulin-bound T (non-SHBG-bound T), is biologically important. To examine the potential exposure of peripheral tissues to T in normal prepubertal boys, we determined the distribution of serum T into SHBG-bound, HSA-bound, non-SHBG-bound, and free fractions in 80 normal males, aged 0.5-14 yr, all at Tanner stage I of sexual development. A model assuming equilibrium between free T and T bound to 2-binding proteins (HSA and SHBG) was used. A computer program, using as constants the SHBG-T and HSA-T affinity constants and the serum HSA concentration and as variables the serum SHBG and total T concentrations, was used to calculate SHBG-bound T, HSA-bound T, non-SHBG-bound T, and free T. Serum total T increased 2.6-fold from 0.5 to 14 yr, whereas non-SHBG-bound T, HSA-bound T, and free T increased 8- to 9-fold during the same period. On the other hand, SHBG-bound T increased only 1.9-fold. Expressed as a function of serum total T, non-SHBG-bound T increased from 6.6% to 30.4%, the relative increment being greater for HSA-bound than for free T. We conclude that with advancing age, there is a progressive increase in the exposure of all tissues to T in normal prepubertal boys. At the level of the central nervous system, this increase in serum bioavailable T could induce maturative changes in brain cells that result in the onset of puberty in normal boys.

Progressive decrease in serum sex hormone-binding globulin from infancy to late prepuberty in boys.

Serum Sex hormone-binding globulin (SHBG) levels and affinity constant (Ka) of SHBG-dihydrotestosterone association were determined in 91 boys, aged 3 months to 15 yr, all at Tanner stage I of pubertal development. A gradual decrease in serum SHBG as a function of age was found in spite of unchanged serum testosterone levels. Ka values at different ages were not significantly different. Since steroids bound to SHBG are not transported into most tissues, particularly brain, a decrease in SHBG will have the effect of increasing tissue entrance of non-SHBG-bound sex hormones despite unchanged plasma concentrations. We speculate that the gradually increasing androgen and estrogen milieu of the brain created by this mechanism might be of physiological significance in triggering the onset of puberty.

Progressive increase in nonsex hormone-binding globulin-bound testosterone and estradiol from infancy to late prepuberty in girls.

We previously reported that serum sex hormone-binding globulin (SHBG) decreases and serum non-SHBG-bound testosterone (T) increases with age in normal prepubertal boys from infancy to late prepuberty. In this study we measured serum SHBG, T, estradiol (E2), and dehydroepiandrosterone sulfate (DS), and we calculated serum non-SHBG-bound T and E2 and free T and E2 in 22 normal prepubertal girls, aged 1-9.4 yr. The girls were divided into 3 groups of different ages: group A, 1.7 +/- 1.0 (mean +/- SE) yr; group B, 4.6 +/- 0.8 yr; and group C, 7.3 +/- 0.8 yr. In group C, mean serum SHBG level was lower, and serum T, non-SHBG-bound T, free T, DS, total E2, non-SHBG-bound E2, and free E2 were higher than in group A or B. Furthermore, a negative correlation was found between serum SHBG and age [y(nmol/L) = 144 - 9.07 x (yr); r = 0.57; P less than 0.001], while positive correlations were found between non-SHBG-bound T and age [y(nmol/L = 0.043 + 0.023 x (yr); r = 0.68; P less than 0.001], non-SHBG-bound E2 and age [y(pmol/L) = 0.69 + 2.82 x (yr); r = 0.6; P less than 0.001], and DS and age [y(nmol/L) = 25.2 + 63.8 x (yr); r = 0.59, P less than 0.001]. In a group of 19 normal age-matched prepubertal boys, we also found a significant correlation between non-SHBG-bound E2 and age. Since non-SHBG-bound sex hormone levels are good indicators of tissue available sex hormones, we conclude that in prepubertal girls, there is a progressive increase in the exposure of peripheral tissues to T and E2 with advancing age. Since sex hormones enhance tissue maturation, these increments might play a role in the somatic and psychic development of girls before the onset of the clinical signs of puberty.

Sex hormone-binding globulin response to human chorionic gonadotropin stimulation in children with cryptorchidism, anorchia, male pseudohermaphroditism, and micropenis.

Sex hormone-binding globulin serum concentrations (SHBG) were measured before and after a 5-day hCG stimulation test in 11 prepubertal boys with cryptorchidism, 6 with anorchia, 5 with male pseudohermaphroditism, and 5 with micropenis. Cryptorchid boys had decreased SHBG levels after hCG, by 55 +/- 17% (mean +/- SE) of the basal concentration. Patients with anorchia, who did not show an elevation in serum androgens, did not have decreased SHBG concentrations. Four of the 5 patients with male pseudohermaphroditism had an adequate elevation of serum androgens, did not have decreased SHBG concentrations. Four of the 5 patients with male pseudohermaphroditism had an adequate elevation of serum androgens after hCG, but in only 3 of them did SHBG decrease. None of the 5 patients with micropenis had decreased serum SHBG levels despite normal increments in serum androgens. The administration of a long-acting preparation of testosterone to sexually infantile subjects produce a similar decrease in the SHBG concentration. This change in SHBG concentration after hCG or testosterone in prepubertal boys could be used as a convenient test of biological response to androgens.

High serum sex hormone-binding globulin (SHBG) and low serum non-SHBG-bound testosterone in boys with idiopathic hypopituitarism: effect of recombinant human growth hormone treatment.

We measured serum sex hormone-binding globulin (SHBG), total testosterone (T), non-SHBG-bound T, albumin-bound T, free T, and SHBG-bound T in 19 prepubertal boys with hypopituitarism. Serum SHBG decreased with age with a slope similar to that in 91 normal prepubertal boys at higher level, and therefore, it reached similar values at a later age. Serum SHBG was significantly higher in hypopituitary prepubertal boys [mean, 123 +/- 12 (+/- SE) nmol/L] than in normal prepubertal boys (76 +/- 4; P less than 0.001) despite the fact that their mean age was also higher (10.0 +/- 4 vs. 7.1 +/- 4.1 yr; P less than 0.001). In 4 boys with isolated hypogonadotropic hypogonadism (Kallman's syndrome), aged 15.6 +/- 1.5 yr, serum SHBG was 21 +/- 14 nmol/L, a value below the 95% confidence limit of the regression line in GH-deficient boys. The affinity constants of association of the SHBG-DHT complex were similar in hypopituitary and normal boys. Eleven of the 19 hypopituitary boys (mean chronological age, 8.3 +/- 2.5 yr; mean bone age, 4.1 +/- 2.1 yr) were treated with recombinant hGH (0.5 U/kg BW.week) for 1 yr. Their mean serum SHBG level before treatment was 154 +/- 14 nmol/L, and it decreased gradually to 106 +/- 5 nmol/L (P less than 0.01) after 12 months of treatment. The tendency toward normalization of serum SHBG during treatment suggested that GH deficiency was responsible for the high serum SHBG levels. Serum SHBG correlated negatively with age in both treated hypopituitary and normal boys, but the slope of the regression line was significantly steeper in treated hypopituitary boys (P less than 0.01). On the other hand, the mean serum non-SHBG-bound T level was 0.10 +/- 0.02 (+/- SE) nmol/L in hypopituitary boys, significantly lower than that in normal boys (0.21 +/- 0.02 nmol/L; P less than 0.02). Since serum total T concentrations were similar in the two groups, the higher serum SHBG concentration resulted in lower serum bioavailable T levels in the hypopituitary boys. These changes might explain the poor response to T treatment reported in GH-deficient patients. The lower serum non-SHBG-bound T concentrations in the GH-deficient boys suggest there may be delayed exposure of central nervous system structures to increased levels of sex hormones, which, in turn, may slow body maturation. This mechanism might play a role in the delay of puberty that occurs in patients with isolated GH deficiency.

Serum sex hormone-binding globulin and serum nonsex hormone-binding globulin-bound testosterone fractions in prepubertal boys with chronic renal failure.

We had previously reported that serum sex hormone binding-globulin (SHBG) decreases and serum non-SHBG-bound testosterone (T) and free T increase significantly from infancy to late prepuberty in normal prepubertal children of both sexes. We had also shown an age-related delay in these changes in hypopituitary boys, which was reversed by GH treatment. Stunted growth and delayed puberty are conspicuous features of chronic renal failure (CRF). As another model of delay of growth and development, serum SHBG and serum T fractions were determined in 13 boys with CRF on chronic dialysis. In CRF, mean serum SHBG was significantly higher (99.1 +/- 68.9 nmol/L; P less than 0.05) than in 31 control (C) children of similar ages (66.2 +/- 34.9 nmol/L), while serum non-SHBG-bound T and free T were significantly lower (0.16 +/- 0.12 in CRF vs. 0.24 +/- 0.12 in C and 0.010 +/- 0.005 in CRF vs. 0.016 +/- 0.01 in C, respectively). On the other hand, serum total T (1.31 +/- 0.88 in CRF vs. 1.08 +/- 0.56 in C) and serum insulin-like growth factor-I (IGF-I; 1.06 +/- 0.74 in CRF vs. 1.35 +/- 1.70 in C) were not significantly different. A significant negative correlation between serum SHBG and chronological age as well as a significant positive correlation between serum non-SHBG-bound T and chronological age were found. For a given age, serum SHBG was higher, while serum non-SHBG-bound T was lower in patients with CRF (by analysis of covariance, P less than 0.01). It is postulated that, as has been proposed for hypopituitary boys, this delayed increment in serum T fractions could be responsible for the delay in the onset of puberty reported in CRF. It is known that GH decreases serum SHBG, acting on hepatic cells either directly or through the action of IGF-I. Since it has been suggested that patients with CRF have peripheral resistance to GH or IGF-I, the high levels of SHBG that we have detected in prepubertal boys with CRF could be taken as an additional evidence of this biological resistance.

Secretion of inhibin B by human prepubertal testicular cells in culture.

OBJECTIVE: Inhibin B is a secretory product of Sertoli cells of the human testis. It has been reported that serum levels of inhibin B in infant boys, peaking at 3 months of age, exceed levels in adult men. The aim of this study was to evaluate inhibin B secretion in primary prepubertal mixed testicular cell cultures, prepared from testes collected at necropsy. DESIGN AND METHODS: Cell cultures were divided into three age groups on the basis of differences in testicular histology: group 1 (n = 7), 1- to 10-day-old newborns, group 2 (n = 7), 1- to 9-month-old infants, and group 3 (n = 8), 12- to 84-month-old children. Cells were maintained in culture for 6 days, harvested and counted. In some samples, during the last 4 days, cells were stimulated with 10ng/ml highly purified human (h) LH (n = 9), 2 ng/ml recombinant human (rh) FSH (n = 9) or 50 ng/ml rhGH (n = 4). On day 6, the secretion of inhibin B and testosterone into the medium was estimated in triplicate. Inhibin B was determined by ELISA and testosterone by RIA. RESULTS: Median (range) inhibin B secretion was 465 (225-1007), 275 (107-298), and 58 (15-184) pg/million cells.24h in groups 1, 2 and 3 respectively. A logarithmic transformation of these values was performed to normalize data. Mean+/-s.d. of transformed inhibin B secretion in group 1 was significantly higher than in group 2 or 3 (P<0.005) and the values for groups 1 and 2 were significantly higher than that for group 3 (P< 0.005). No significant correlation between testosterone and inhibin B secretion into the medium was found when the 22 culture samples were analyzed as a whole. Inhibin B secretion was significantly increased after stimulation with highly purified hLH, rhFSH and rhGH (P < 0.05) and a significant positive correlation between inhibin B and testosterone was found under both hLH and rhFSH stimulation. CONCLUSIONS: It is concluded that cells collected from newborns have the highest capacity to secrete inhibin B in vitro, and that this capacity decreases with age during the first years of life. Since no data are available on serum inhibin levels in newborns, it is possible that concentrations at 3 months of age do not represent a post-natal peak but a declining level of high newborn values. As expected, FSH stimulated inhibin B secretion in culture. LH stimulation was probably mediated by paracrine factors secreted by interstitial cells. Finally, our results add new evidence of the involvement of GH in testicular maturation.

Human prepubertal testicular cells in culture: steroidogenic capacity, paracrine and hormone control.

The neonatal human Leydig cell undergoes a transient period of activation during the first months of life. The biological significance of this activation is unknown. Furthermore, little is known about the hormonal regulation of this biological process, even though it coincides with an elevation of LH levels in serum. In order to study the function of human prepubertal testicular culture cells, obtained during the neonatal period, a method for maintaining primary culture cells (isolated from testes collected at necropsy) in culture was developed. Within 24 h after death, testes were collected from 1-36-month-old subjects. Subjects were divided into two age groups, based on the presence or absence of fetal Leydig cells: 1-7-month-old infants (group 1) and 12-36-month-old children (group 2). Testes were digested with collagenase, and cells were seeded in multi-well dishes. Cells were grown in serum-free conditioned media supplemented with 5 mg/l vitamin C, 0.2 IU/l vitamin E and 10% fetal bovine serum for 2 days. Cells were then grown for an additional 4 days in serum-free media in the presence or absence of hLH (40 IU/l), hCG (135 IU/l), rh FSH (1.5 IU/l), rhGH (0.12 IU/l) or insulin (0.9 mumol/l). Concentrations of steroids in media were determined by RIA on day 6 of culture. In basal conditions cells of group 1 (n = 11) secreted more testosterone, androstendione, 17-hydroxyprogesterone, progesterone and dehydroepiandrosterone (mean +/- SE: 6.76 +/- 1.86, 7.37 +/- 1.82, 61.9 +/- 1.86, 5.75 +/- 1.74 and 8.51 +/- 3.23 pmol/10(6) cells/24 h, respectively) than cells of group 2 (n = 5) (2.95 +/- 1.15, 1.50 +/- 2.75, 1.44 +/- 2.75, 0.78 +/- 1.74 and 3.23 +/- 1.32, respectively). Under hLH stimulation, cells of group 1 increased testosterone, androstendione and 17-hydroxyprogesterone secretions (to 38.2 +/- 0.89, 13.5 +/- 1.17 and 51.7 +/- 3.23), while progesterone secretion remained unchanged (2.82 +/- 1.20). Cell response to rhFSH and rhGH was similar to that of hLH. On the other hand, medium collected from cultures of cells isolated from a Sertoli cell tumor was able to stimulate testosterone secretion in subcultures of control testicular cells in a way similar to that of hCG. In conclusion, (1) these prepubertal human testicular cells can be maintained in primary culture for several days keeping their in vivo steroidogenic potential; (2) cells isolated from young infants can respond to hLH in culture; (3) response to rhFSH is probably mediated by a paracrine factor; (4) response to rhGH is observed in the absence of gonadotropins.(ABSTRACT TRUNCATED AT 400 WORDS)

Testicular steroid biosynthesis in a boy with a large cell calcifying Sertoli cell tumor producing prepubertal gynecomastia.

A study of a large cell calcifying Sertoli cell tumor of the testis associated with bilateral gynecomastia in an 8-year-old boy is presented. Macroscopically, the two testes showed multiple, large, and hard calcified nodules. Histology revealed clusters or cords of tumor cells with foci of calcifications as well as evidences, in the adjacent testicular parenchyma, of initiation of gonadal development, such as early signs of spermatogenesis and sparse Leydig cell differentiation. In vivo, serum hormone studies showed gonadotropin-independent gonadal activity. After orchidectomy two macroscopically distinct fractions of the removed testes, tumoral and extratumoral, were processed separately for cell isolation and culture. The secretion of testosterone, androstenedione, and 17-hydroxyprogesterone to the medium on day 6 of culture showed that steroidogenesis in cells of the extratumoral fraction was more active than in the tumoral fraction. On the other hand, tumoral fraction cells showed much higher aromatase activity than extratumoral cells. Furthermore, conditioned medium of tumoral fraction cells was able to stimulate testosterone secretion when it was added to subcultures of testicular cells isolated from a control subject. It is postulated that tumoral cells might have stimulated neighboring interstitial cells to differentiate into Leydig cells and to secrete androgens, which in turn might have been aromatized to estrogens by tumoral cells.

Mutations of the steroid 21-hydroxylase gene in an Argentinian population of 36 patients with classical congenital adrenal hyperplasia.

In several studies carried out in USA and Europe, gene deletions, large gene conversions and six point mutations accounted for over 90% of the mutated alleles reported in classical congenital hyperplasia (CAH). In order to know the relative frequencies of mutations in a Latin-American population, the CYP21 active gene was analyzed in 42 patients with CAH belonging to 36 families attending two Argentinian clinics. The salt wasting form was diagnosed in 24 index cases and the simple virilized form in 12. When available, parents were also studied. DNA was extracted from peripheral blood leukocytes and specific PCR amplification of four different fragments of the CYP21 gene was carried out, followed by electrophoresis of the amplified product. The four fragments include segments of the gene containing the six most frequently reported abnormalities in classical CAH: IN2, EX3, R356W, cluster EX6 and I172N. Point mutations were studied by allelic specific oligonucleotide hybridization; Q318X was studied by digestion of the PCR product with PsT1 restriction enzyme and electrophoresis on 6% non-denaturing polyacrylamide gels. Deletions and macroconversions as well as confirmation of homozygote point mutations were studied by Southern blotting. Percentage distribution of abnormalities was as follows: deletion/macroconversion 18, IN2 18, I172N 15.3, Q318X 13.8, R356W 5.5, EX3 2.7, cluster EX6 0, not characterized 26.7. The complete genotype could be determined in 20 families while in 12 additional ones, the mutation was detected in one allele. Deletion/macroconversion, IN2, EX3 and Q318X were detected more frequently in salt wasting patients while I172N and R356W were found in simple virilized patients. However, genotype was not always concordant with phenotype. It is concluded that there are differences in the frequency of several gene mutations and in that of deletion/macroconversion between this Latin-American population and several reported American and European populations. In particular the percentage of deletion/macroconversion, IN2, EX3 and cluster EX6 was lower while I172N was higher in our Latin-American population. Furthermore the frequency of mutations not characterized was larger. This information is useful to delineate appropriate strategies for prenatal diagnosis in this particular population.

Expression of the aromatase gene in the human prepubertal testis.

It has been proposed that estrogens might play a negative feedback role in the local regulation of androgen biosynthesis in the testis. Although aromatase has been reported to be present in human adult Leydig cells, CYP19 gene expression in the human prepubertal testis has not been studied. Human prepubertal testicular tissue was obtained from 12 testes collected at necropsy. Ages ranged from 0.07 to 7 years, but 7 of the 12 subjects were younger than 3 months old. Tissue mRNA was subjected to RT-PCR analysis by two methods. Cytochrome P450arom mRNA was detected by non-radioactive RT-PCR in five of the 12 prepubertal testes collected from 0.05-7 year-old subjects, and in one testis collected from a 15 year-old pubertal control. Four of these five prepubertal samples belonged to the youngest infant group. Using a more sensitive, radioactive RT-PCR, aromatase mRNA was detected in all prepubertal testes. This study shows that the CYP19 gene is expressed in the prepubertal human testis including the period of early postnatal activation. It is possible that estrogens may have a role in prepubertal males during this period.

[Role of 3 beta hydroxysteroid dehydrogenase (3 beta HSD) in human adrenal androgen synthesis]. / Implicancias de la enzima 3 beta hidroxiesteroide dehidrogenasa (3 beta HSD) en la síntesis de andrógenos en la adrenal humana.

Adrenarche is the maturational increase of adrenal androgens that takes place in 6-8 year old children. In order to study the role of 3 beta HSD in the regulation of the synthesis of human adrenal androgens, the abundance of 3 beta HSD mRNA (Dot Blot and semiquantitative RT-PCR) was measured in 11 human prepubertal and early pubertal adrenal tissues. Subjects were divided in 2 age groups (Gr): Gr1, < 8 years (y) old (n = 6, range 0.1-2.5) and Gr2, > or = 8 y old (n = 5, range 8.0-13.0). Tissue from one adrenal tumor with Cushing's syndrome (TSC) and 2 virilizing adrenal tumors (TV), as well as adrenal cells prepared from the TSC and from 1 TV were also studied. They were maintained in culture for 3 days in basal conditions (BC) and under ACTH and IGF-1 stimulation. mRNA in Gr1 was higher than in Gr2 (Dot blot: 4.65 +/- 2.70 and 0.28 +/- 0.27 AU, p = 0.006; RT-PCR: 21.5 +/- 12.5 and 6.77 +/- 3.78 AU, p = 0.039, respectively). 3 beta HSD mRNA in TSC (8.74 +/- 1.74) was higher than in the 2 TVs (0.47 +/- 0.02 and 0.87 +/- 0.08) p = 0.001. In TSC cells, basal mRNA (0.82 +/- 0.10) decreased under ACTH (0.55 +/- 0.06), p = 0.005, and increased under IGF-1 (2.36 +/- 0.07), p = 0.006. No changes were observed in TV cells. On day 3, TV cells in BC secreted 1170.0 +/- 210.0 and 335.0 +/- 29.0 pmol/10(6) cells in 24 hs of DHEAS and androstenedione, while TSC cells secreted 17.1 +/- 3.5 and 73.7 +/- 11.7, respectively. Values increased under ACTH in TV cells (2006.0 +/- 360.0 and 525.0 +/- 76.0) and in TSC cells (29.8 +/- 5.4 and 366.8 +/- 129) p < 0.05, but they decreased under IGF-1 only in TSC cells (7.9 +/- 2.4 and 43.7 +/- 6.1) p < 0.05. These data support the hypothesis that human adrenarche could be secondary to a decrease of 3 beta HSD mRNA. Our finding that when 3 beta HSD mRNA decreases androgen secretion increases (ACTH) and when 3 beta HSD mRNA increases androgen secretion decreases (IGF-1), strongly suggests that 3 beta HSD has a modulatory role in adrenal androgen steroidogenesis.

[Molecular analysis of the most frequent mutations associated with congenital adrenal hyperplasia secondary to 21-hydroxylase enzyme deficiency]. / Análisis molecular de las mutaciones más frecuentes asociadas a la hiperplasia suprarrenal congénita por déficit de la enzima 21 hidroxilasa.

Most cases (90%) of congenital adrenal hyperplasia (CAH) are secondary to steroid 21-hydroxylase enzyme deficiency (P450c21). In human, the P450c21 gene (CYP21B) is present along with a non functional pseudogene (CYP21A). These genes, located in chromosome 6, present a sequence homology of 98%. This high homology and the complexity of this gene locus brings about considerable difficulties in its molecular analysis and in the interpretation of the results. The aim of the present study was to elaborate an adequate strategy for the analysis of the most frequent mutations described in the CYP21B gene. A total of 77 patients with clinical and biochemical diagnosis of CAH secondary to P450c21 enzyme deficiency, as well as 170 unaffected relatives, were studied. They belonged to 73 unrelated families (146 chromosomes). The strategy allowed for the differentiation of patients with homozygous point mutations (PM), with PM in one allele and deletions, conversions, Ex3 or Cluster Ex6 PM in the other, even though parents were not always available for the study. Furthermore, it allowed for the discrimination of heterozygous deletions or conversions of the CYP21B gene from duplications of the non functional gene CYP21A, as well as CYP21B and A deletions from normal copies of the two genes. An exhaustive molecular analysis of this gene is necessary for an adequate characterization of the alterations present in this locus.

Changes in the distribution of testosterone and estradiol serum fractions in hirsute women after administration of L-thyroxine.

The effect of 1-thyroxine treatment on the distribution of testosterone and estradiol serum fractions was studied in 10 normal weight hirsute women with ages ranging from 18 to 25 years. Six of the patients had menstrual irregularities. Treatment consisted in one month of thyroxine (1-T4) at a dose of 2 micrograms/Kg body weight. Serum sex hormone binding globulin (SHBG) was measured by saturation analysis and serum testosterone (T), estradiol (E2), androstenedione (delta 4-A), dehydroepiandrosterone sulphate (DHAS), LH, FSH, PRL, T4, T3, by RIA. Serum T and E2 fractions were calculated by an equation derived from the law of mass action. Before and after T4 treatment serum SHBG was, respectively, (X +/- ES) 30.3 +/- 3.8 and 45.4 +/- 4.6 nmol/l (p less than 0.001), non-SHBG-bound T 25.7 +/- 8.1 and 18.8 +/- 6 ng/dl (p less than 0.001), free T 1.09 +/- 0.2 and 0.8 +/- 0.5 ng/dl (p less than 0.01), non-SHBG-bound E2 40.9 +/- 13.7 and 19.2 +/- 2.4 pg/ml (p less than 0.001). No significant changes were observed in total T and E2 or free E2. Serum DHAS and delta 4-A decreased significantly after treatment from 3977 +/- 601 to 3480 +/- 250 ng/dl and from 4.7 +/- 0.61 to 3.34 +/- 0.34 ng/ml respectively (p less than 0.001). Serum basal levels of FSH, LH, PRL, T4, T3 and TSH were normal. A significant increase of serum T4 was observed after treatment from 8.4 +/- 0.4 to 11.7 +/- 1.1 ng/dl (p less than 0.01). However, the post-treatment value was within the limits of the normal range.(ABSTRACT TRUNCATED AT 250 WORDS)

Estudio de la expresión del receptor de vitamina D en la ontogenia del testículo y en tumores de células de leydig: posible interacción con el sistema histaminérgico

RESUMEN La vitamina D (VD), un esteroide pleiotrópico, ha sido relacionada con la función reproductiva masculina, pero aún no se ha estudiado la expresión de su receptor (RVD) en el desarrollo testicular. RVD regula la expresión de componentes del sistema histaminérgico, y la histamina (HA) modula la esteroidogénesis en células de Leydig (CL). Se ha relacionado a la deficiencia de VD con múltiples patologías, entre ellas cáncer. Los tumores de células de Leydig (TCL) son los más frecuentes del intersticio testicular, y al malignizar no responden a radio/quimioterapia. VD fue descripta como tratamiento para varios tumores, pero se desconoce su aplicación en TCL. Por lo expuesto, hemos estudiado la expresión de RVD en la ontogenia de testículo de rata, evaluando su correlación con los niveles de testosterona séricos (T) y el contenido de HA; y además evaluamos la expresión de RVD en testículo humano fetal, neonatal, prepuberal, TCL e hiperplasia de CL. En testículo de rata, se observó un aumento en la expresión de RVD en CL con la edad, en línea con el incremento de T, y en contraposición con la disminución del contenido de HA, lo cual fue consistente con la reducción en los niveles de la enzima que cataliza su síntesis, HDC. Esto sugiere que la VD podría ejercer una función en el desarrollo testicular normal, ya sea en forma directa sobre las CL o mediante la regulación de la expresión de componentes del sistema histaminérgico (HDC y/o receptores de HA). Por su parte, el TCL humano presentó sobreexpresión de RVD y HDC. Considerando que las hormonas esteroideas se encuentran aumentadas en esta patología y funcionan como factores de crecimiento, si el calcitriol pudiera modular la esteroidogénesis podría tener una aplicación terapéutica.

ABSTRACT Vitamin D (VD) is a steroid hormone traditionally related to bone health. However, several authors have associated VD with reproduction and steroidogenesis in males. The presence ofVD receptor (VDR) and the enzymes involved in its activation had been reported in several cell types of the testes. Until now, nobody has studied RVD expression during testicular development. In addition, VDR in association with its co-activators or co-repressors, regulates the expression of several genes, including those related to the histaminergic system. Previously, we demonstrated that histamine (HA) can modulate steroidogenesis in Leydig cells (LC) in a concentration-dependent manner. Also, we observed a decrease in the endogenous HA content, consistent with the previously described decrease of HDC (histidine decarboxylase, the enzyme responsible of HA synthesis) levels, during LC ontogeny. Epidemiologic studies strongly suggest that a relationship exists between VD deficiency and multiple pathologies, particularly cancer. Leydig cell tumors (LCT) are rare endocrine tumors ofunknown etiology, which originate in the testicular interstitium. The incidence worldwide is 1-3% in adults and 4% in prepubertal boys, but recent publications indicate that these figures have been increasing. While usually benign, approximately 10% of LCT in adults become malignant and do not respond to chemo or radiotherapy. It is imperative to deeply investigate the biology of LCT, to identify new therapeutic targets. The potential role of calcitriol (1a,25(OH)2-vitamin-D3) in cancer treatment has been described for several types of tumors, but it remains unexplored in LCT. Thus, as a first step, it is worth evaluating VDR expression in LCT.In view of the aforecited evidence, herein we studied VDR expression during the rat testicular ontogeny, evaluating a possible correlation withserum testosterone (T) levels in blood, endogenous levels of HA and the previously described HDC expression levels. We also analized VDR expression in human testes corresponding to three different stages of development (fetal, neonatal andjuvenile), in LCT and in LC hyperplasia. Methods: Rat testes of different ages (7, 21, 35, 90 y 240 days), human fetal, neonatal and pre pubertal testes, a human LCT and a human LC hyperplasia; were used for detection of VDR by immunohistochemistry. Results: In the rat testes, VDR expression increased with age in LC, in line with the increase in serum testosterone; and in contrast with the decrease in the endogenous content of HA and HDC levels. Likewise, we detected an increase in VDR expression with age in the human testes samples. LCT presentedVDR and HDC overexpression. We also detected VDR in LC hyperplasia. Conclusions: Given that VDR testicular expression increases with age in LC, as well as testosterone serum levels, it is reasonable to speculate thatVD may play a role in normal testicular development, either acting directly on LC or by regulating one of more components of the histaminergic system (HDC or HA receptors). Considering that VDR is overexpressed in LCT, and that steroids are increased in this pathology (and act like growth factors); if calcitriol could modulate steroidogenesis, it could have a therapeutic role.

A pharmacogenomic approach to the treatment of children with GH deficiency or Turner syndrome.

OBJECTIVE: Individual sensitivity to recombinant human GH (r-hGH) is variable. Identification of genetic factors contributing to this variability has potential use for individualization of treatment. The objective of this study was to identify genetic markers and gene expression profiles associated with growth response on r-hGH therapy in treatment-naïve, prepubertal children with GH deficiency (GHD) or Turner syndrome (TS). DESIGN: A prospective, multicenter, international, open-label pharmacogenomic study. METHODS: The associations of genotypes in 103 growth- and metabolism-related genes and baseline gene expression profiles with growth response to r-hGH (cm/year) over the first year were evaluated. Genotype associations were assessed with growth response as a continuous variable and as a categorical variable divided into quartiles. RESULTS: Eleven genes in GHD and ten in TS, with two overlapping between conditions, were significantly associated with growth response either as a continuous variable (seven in GHD, two in TS) or as a categorical variable (four more in GHD, eight more in TS). For example, in GHD, GRB10 was associated with high response (≥ Q3; P=0.0012), while SOS2 was associated with low response (≤ Q1; P=0.006), while in TS, LHX4 was associated with high response (P=0.0003) and PTPN1 with low response (P=0.0037). Differences in expression were identified for one of the growth response-associated genes in GHD (AKT1) and for two in TS (KRAS and MYOD1). CONCLUSIONS: Carriage of specific growth-related genetic markers is associated with growth response in GHD and TS. These findings indicate that pharmacogenomics could have a role in individualized management of childhood growth disorders.