A meta-analytic investigation of linkage and association of common leptin receptor (LEPR) polymorphisms with body mass index and waist circumference.

METHODS: We analyzed data pooled from nine studies on the human leptin receptor (LEPR) gene for the association of three alleles (K109R, Q223R and K656N) of LEPR with body mass index (BMI; kg/m(2)) and waist circumference (WC). A total of 3263 related and unrelated subjects from diverse ethnic backgrounds including African-American, Caucasian, Danish, Finnish, French Canadian and Nigerian were studied. We tested effects of individual alleles, joint effects of alleles at multiple loci, epistatic effects among alleles at different loci, effect modification by age, sex, diabetes and ethnicity, and pleiotropic genotype effects on BMI and WC. RESULTS: We found that none of the effects were significant at the 0.05 level. Heterogeneity tests showed that the variations of the non-significant effects are within the range of sampling variation. CONCLUSIONS: We conclude that, although certain genotypic effects could be population-specific, there was no statistically compelling evidence that any of the three LEPR alleles is associated with BMI or WC in the overall population.

Pooling analysis of genetic data: the association of leptin receptor (LEPR) polymorphisms with variables related to human adiposity.

Analysis of raw pooled data from distinct studies of a single question generates a single statistical conclusion with greater power and precision than conventional metaanalysis based on within-study estimates. However, conducting analyses with pooled genetic data, in particular, is a daunting task that raises important statistical issues. In the process of analyzing data pooled from nine studies on the human leptin receptor (LEPR) gene for the association of three alleles (K109R, Q223R, and K656N) of LEPR with body mass index (BMI; kilograms divided by the square of the height in meters) and waist circumference (WC), we encountered the following methodological challenges: data on relatives, missing data, multivariate analysis, multiallele analysis at multiple loci, heterogeneity, and epistasis. We propose herein statistical methods and procedures to deal with such issues. With a total of 3263 related and unrelated subjects from diverse ethnic backgrounds such as African-American, Caucasian, Danish, Finnish, French-Canadian, and Nigerian, we tested effects of individual alleles; joint effects of alleles at multiple loci; epistatic effects among alleles at different loci; effect modification by age, sex, diabetes, and ethnicity; and pleiotropic genotype effects on BMI and WC. The statistical methodologies were applied, before and after multiple imputation of missing observations, to pooled data as well as to individual data sets for estimates from each study, the latter leading to a metaanalysis. The results from the metaanalysis and the pooling analysis showed that none of the effects were significant at the 0.05 level of significance. Heterogeneity tests showed that the variations of the nonsignificant effects are within the range of sampling variation. Although certain genotypic effects could be population specific, there was no statistically compelling evidence that any of the three LEPR alleles is associated with BMI or waist circumference in the general population.

Sympathetic inhibition, leptin, and uncoupling protein subtype expression in normal fasting rats.

To further investigate neural effects on leptin and uncoupling proteins (UCPs), we studied in vivo perturbations intended to block adrenergic input to peripheral tissues. We examined plasma leptin, leptin mRNA, and adipose and muscle UCP subtype mRNA in rats treated with alpha-methyl-p-tyrosine methyl ester (AMPT-ME), which inhibits catecholamine synthesis and 6-hydroxydopamine (6HDA), which is toxic to catecholinergic nerve terminals but, unlike AMPT-ME, does not enter the central nervous system. Intraperitoneal AMPT-ME, 250 mg/kg, was administered at 1800 and 0700 the following day, and rats were killed at 1200-1400. All rats were fasted with free access to water during this time. Intraperitoneal AMPT-ME increased plasma leptin by 15-fold, increased interscapular brown adipose tissue (IBAT) and epididymal fat leptin mRNA by 2- to 2.5-fold, and also increased plasma insulin and glucose concentrations. Intraperitoneal AMPT-ME decreased IBAT UCP-3 mRNA to 40% of control, while it increased epididymal adipose UCP-3 mRNA approximately twofold. Intravenous AMPT-ME, 250 mg/kg, administered to conscious rats for 5 h decreased lumbar sympathetic nerve activity, increased plasma leptin (5.89 +/- 1.43 compared with 2.75 +/- 0.31 ng/ml in vehicle-treated rats, n = 7, P < 0.05), and decreased cardiac rate with no sustained change in blood pressure. Intraperitoneal 6HDA, 100 mg/kg, as a single dose at 1800, increased plasma leptin approximately twofold after 18-20 h, increased IBAT (but not epididymal fat) leptin mRNA by two- to threefold, and decreased IBAT UCP-3 mRNA to 30-40% of control. Neither AMPT-ME nor 6HDA significantly altered mRNA encoding gastrocnemius muscle UCP-3, IBAT UCP-1, or IBAT and epididymal UCP-2. In summary, AMPT-ME and 6HDA increased plasma leptin and upregulated leptin mRNA expression. AMPT-ME also resulted in complex tissue and subtype-specific modulation of adipose UCP mRNA. These data are consistent with interaction between leptin and sympathetic nerve activity (SNA) in regulation of fat cell energy utilization. However, the in vivo modulation of leptin and UCPs appears complex and, beyond a causal effect of SNA per se, may depend on concurrent changes in plasma insulin, glucose, and circulatory hemodynamics.

Fasting and leptin modulate adipose and muscle uncoupling protein: divergent effects between messenger ribonucleic acid and protein expression.

Leptin is believed to act through hypothalamic centers to decrease appetite and increase energy utilization, in part through enhanced thermogenesis. In this study, we examined the effects of fasting for 2 days and exogenous s.c. leptin, 200 microg every 8 h for 2 days, on the regulation of uncoupling protein (UCP) subtypes in brown adipose tissue (BAT) and gastrocnemius muscle. Northern blot analysis (UCP-1) and ribonuclease protection (UCP-2 and 3) were used for quantitative messenger RNA (mRNA) analysis, and specific antibodies were used to measure UCP-1 and UCP-3 total protein expression. Leptin, compared with vehicle, did not alter BAT UCP-1 or UCP-3 mRNA or protein expression when administered to normal ad libitum fed rats. Fasting significantly decreased BAT UCP-1 and UCP-3 mRNA expression, to 31% and 30% of ad libitum fed controls, respectively, effects which were prevented by administration of leptin to fasted rats. Fasting also significantly decreased BAT UCP-1 protein expression, to 67% of control; however, that effect was not prevented by leptin treatment. Fasting also decreased BAT UCP-3 protein, to 85% of control, an effect that was not statistically significant. Fasting, with or without leptin administration, did not affect BAT UCP-2 mRNA; however, leptin administration to ad libitum fed rats significantly increased BAT UCP-2 mRNA, to 138% of control. Fasting significantly enhanced gastrocnemius muscle UCP-3 mRNA (411% of control) and protein expression (168% of control), whereas leptin administration to fasted rats did not alter either of these effects. In summary, UCP subtype mRNA and protein are regulated in tissue- and subtype-specific fashion by leptin and food restriction. Under certain conditions, the effects of these perturbations on UCP mRNA and protein are discordant.

Clinical and functional effects of mutations in the DAX-1 gene in patients with adrenal hypoplasia congenita.

Adrenal hypoplasia congenita (AHC) is an X-linked disorder caused by mutations in a gene referred to as DAX-1. AHC is characterized by adrenal insufficiency and failure to undergo puberty because of hypogonadotropic hypogonadism. The DAX-1 protein is structurally related to orphan nuclear receptors, although it lacks the characteristic zinc finger DNA-binding domain that is highly conserved in other members of this family. In this report, we describe the clinical features and genetic alterations in six families with AHC. These patients reveal the variable clinical presentation of adrenal insufficiency in AHC and underscore the importance of considering this diagnosis. Nonsense mutations that introduce a stop codon were found in three cases (W171X, W171X, Y399X). Frameshift mutations (405delT, 501delA, and 702delC), each of which resulted in a premature stop codon at amino acid 263, were found in the other three families. Three of these mutations (Y399X, 405delT, 702delC) are novel. Using transient gene expression assays to assess DAX-1 function, these mutations were shown to eliminate the ability of DAX-1 to repress the transcription of genes that are stimulated by a related nuclear receptor, steroidogenic factor-1. These studies reveal the variable clinical presentation of DAX-1 mutations and emphasize the value of genetic testing in boys with primary adrenal insufficiency and suspected X-linked AHC.

Phenotypic evolution of classic 21-hydroxylase deficiency.

We describe a female patient who was diagnosed and treated at birth for a classic form of salt-losing congenital adrenal hyperplasia. At 17 years of age, against medical advice, she discontinued both mineralocorticoid and glucocorticoid replacement with no resulting clinical symptoms other than the occurrence of amenorrhoea. Steroid metabolites revealed significant abnormalities of the renin-angiotensin-aldosterone axis, as well as of pituitary-adrenal function. Analysis of our patient's DNA showed only one deleterious CYP21 mutation, an intron 2 base pair change activating a cryptic splice site. We speculate that expression of this patient's CYP21 genes may be altered by the effects of ageing or by changes in the steroid milieu.

Linkage of congenital isolated adrenocorticotropic hormone deficiency to the corticotropin releasing hormone locus using simple sequence repeat polymorphisms.

Genetic screening techniques using simple sequence repeat polymorphisms were applied to investigate the molecular nature of congenital isolated adrenocorticotropic hormone (ACTH) deficiency. We hypothesize that this rare cause of hypocortisolism shared by a brother and sister with two unaffected sibs and unaffected parents is inherited as an autosomal recessive single gene mutation. Genes involved in the hypothalamic-pituitary axis controlling cortisol sufficiency were investigated for a causal role in this disorder. Southern blotting showed no detectable mutations of the gene encoding pro-opiomelanocortin (POMC), the ACTH precursor. Other candidate genes subsequently considered were those encoding neuroendocrine convertase-1, and neuroendocrine convertase-2 (NEC-1, NEC-2), and corticotropin releasing hormone (CRH). Tests for linkage were performed using polymorphic di- and tetranucleotide simple sequence repeat markers flanking the reported map locations for POMC, NEC-1, NEC-2, and CRH. The chromosomal haplotypes determined by the markers flanking the loci for POMC, NEC-1, and NEC-2 were not compatible with linkage. However, 22 individual markers defining the chromosomal haplotypes flanking CRH were compatible with linkage of the disorder to the immediate area of this gene on chromosome 8. Based on these data, we hypothesize that the ACTH deficiency in this family is due to an abnormality of CRH gene structure or expression. These results illustrate the useful application of high density genetic maps constructed with simple sequence repeat markers for inclusion/exclusion studies of candidate genes in even very small nuclear families segregating for unusual phenotypes.

Constitutive human steroid 21-hydroxylase promoter gene and pseudogene activity in steroidogenic and nonsteroidogenic cells with the luciferase gene as a reporter.

This study was directed toward initial comparison and characterization of the activities of the human steroid 21-hydroxylase gene (CYP21) and pseudogene (CYP21P) promoters. DNA fragments containing the promoter regions of CYP21 and CYP21P were amplified and cloned into promoterless luciferase reporter plasmids either containing or lacking an enhancer element. Cells of the nonsteroidogenic COS-1 cell line, and the steroidogenic Y-1 cell line were transiently transfected with these recombinant plasmids and a beta-galactosidase cotransfection control plasmid. Cellular lysates were analyzed for luciferase and beta-galactosidase activities. In the nonsteroidogenic system, transfectants with either the CYP21 or CYP21P upstream sequence in enhancer containing plasmids showed a 2.3 fold increase (p < .001) in light production over controls. In the steroidogenic Y-1 cell system, these same CYp21 and CYP21P transfectants showed a 14.3 (+/- 0.8) and 5.2 (+/- 0.6) fold increase in luciferase activity respectively (p < .001) Transfections with recombinant reporter plasmids lacking an enhancer produced light emission which was not significantly different than controls. These observations indicate that 1.) one or more of the 35 nucleotide differences between the CYP21 and CYP21P upstream regions alters a DNA recognition site important for transcriptional activation of this gene in steroidogenic cells, 2.) the steroidogenic milieu has a stimulatory effect on both CYP21 and CYP21P promoter activities, and 3.) based on the minimal promoter activity observed in either cell type transfected with constructs lacking an enhancer element, both of these promoter sequences are enhancer dependent under constitutive conditions in both steroidogenic and nonsteroidogenic cells.

The HLA-A3, Cw6,B47,DR7 extended haplotypes in salt losing 21-hydroxylase deficiency and in the Old Order Amish: identical class I antigens and class II alleles with at least two crossover sites in the class III region.

The HLA-B47,DR7 haplotype in congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency contains a deletion of most of the active CYP21 gene and the entire adjacent C4B gene. The C4A gene produces a protein which is electrophoretically C4A but antigenically C4B. In the Old Order Amish, the HLA-B47,DR7 haplotype contains no deletion, but is immunologically identical to the CAH haplotype in both areas flanking the crossover region. We compared some of the genes in the MHC Class II and Class III regions in the Amish and CAH-linked haplotypes to define further the relationships between the two. The complement factor B (Bf) proteins differed, but no Bf RFLPs were identified. The complement factor 2 genes exhibited different BamHI RFLPs. Analyses of the tumor necrosis factor-alpha genes revealed the same NcoI restriction patterns. The RD genes contained microsatellites of the same size. Portions of the MHC Class II DR and DQ, and Class III CYP21 and C4 alleles were sequenced. The exon 2 sequences of DQ2 and DR7 were identical in the two haplotypes. In the Amish haplotype, both CYP21 and C4 gene pairs were present and functionally normal. The CAH haplotype had two sequence crossovers: from CYP21P to CYP21 in the 7th intron, and from C4A to C4B between codons 1106 (exon 26) and 1157 (exon 28). A model is proposed which accounts for the CAH-linked mutant haplotype arising from a nonmutant homologue via three crossings-over.

The human complement C4B/steroid 21-hydroxylase (CYP21) and complement C4A/21-hydroxylase pseudogene (CYP21P) intergenic sequences: comparison and identification of possible regulatory elements.

We determined the 1.8 kb intergenic sequences between the human complement C4B gene and the active steroid 21-hydroxylase gene in two subjects, and between the C4A gene and the steroid 21-hydroxylase pseudogene in one subject. Comparison of these sequences with each other and with published homologues revealed no differences which were unique to either intergenic region. Sequence analysis revealed two copies of an AGGTCA motif in all sequences. This motif is common to steroidogenic enzyme gene promoters and to the response elements for nuclear hormone receptors. Similarities with human enhancers were also found.

Pituitary gigantism caused by growth hormone excess from infancy.

A 2 1/4-year-old boy with pituitary gigantism had a large pituitary macroadenoma. Gross tumor removal has prevented further visual losses, but he has had persistent hypersecretion of growth hormone and prolactin. Treatment with somatostatin analog has decreased both hormone secretion and growth velocity.

Accelerated growth rates in children treated with growth hormone after renal transplantation.

To determine the usefulness of growth hormone treatment among children with renal allografts, we treated nine children with functioning renal transplants who were less than 16 years of age and had poor growth. The nine children, who were aged 12.6 +/- 4.0 years, had (1) heights greater than 2.5 SD less than the mean for age, (2) growth rates less than or equal to 5 cm/yr, and (3) additional growth potential, as assessed by bone age (8.9 +/- 2.8 year). Insulin-like growth factor I, thyrotropin, and thyroid hormone levels were normal for age in all children. Growth hormone treatment increased growth rates from 1.9 +/- 1.1 cm/yr to 7.2 +/- 1.8 cm/yr without accelerating skeletal maturation and without advancing pubertal status. During growth hormone treatment, serum creatinine concentration rose from 140 +/- 50 to 190 +/- 80 mumol/L (1.6 +/- 0.6 to 2.1 +/- 0.9 mg/dl) (p less than 0.05), and creatinine clearances decreased from 0.79 +/- 0.37 to 0.58 +/- 0.30 ml/sec per 1.73 m2 (47 +/- 22 to 35 +/- 18 ml/min per 1.73 m2) (p less than 0.05) but then remained stable. Growth rates of two patients returned to pretreatment rates when growth hormone treatment was discontinued after 5 and 7 months because of increased serum creatinine values. Growth hormone treatment may be useful as adjunctive therapy for increasing growth rates in selected children with renal allografts who have poor growth; however, serum creatinine concentrations should be closely monitored during such treatment.

Congenital adrenal hyperplasia caused by 21-hydroxylase deficiency. Its molecular basis and its remaining therapeutic problems.

This article discusses congenital adrenal hyperplasia (CAH) caused by a deficiency of 21-hydroxylase, which represents 90% of all cases of CAH. As in other genetic disorders of metabolism, the symptoms of CAH are related to both the decrease of the final products of metabolism and the accumulation of precursors that are not normally secreted or that are secreted in only very small amounts. The biochemistry, pathophysiology, treatment, genetics, and long-term follow-up (including fertility and sexual orientation) of both the simple virilizing form and the salt-losing form of 21-hydroxylase deficiency are presented, as well as the possibility of prenatal treatment.

Glucose tolerance in children with renal allografts and effect of growth hormone treatment.

We performed oral glucose tolerance tests (oGTTs) on 15 children who had functioning renal allografts received greater than or equal to 18 months previously, had adequate renal function, and had heights greater than 2.5 SD below the mean height for age. Three of the children had impaired glucose tolerance; their mean glucose levels during the last 2 hours of the oGTT were higher (p less than 0.05) than published control values. Integrated glucose concentrations correlated inversely with the prednisone dose on the first day of an alternate-day dosage schedule (R2 = 0.383) and directly with adiposity (partial R2 = 0.322). The integrated insulin concentration correlated directly with the prednisone dose on day 1 of an alternate-day regimen (R2 = 0.355) and with age (partial R2 = 0.163). In 10 children with renal transplants who had been treated with growth hormone for greater than or equal to 6 months, the mean fasting glucose concentration, integrated glucose concentration, and integrated insulin concentration during the oGTTs obtained after 6 months or 12 months of growth hormone treatment were not significantly different (p greater than 0.05) from values measured before the treatment. We conclude that increased integrated concentrations of both glucose and insulin during oGTTs in children with renal allografts correlate with the dose of prednisone administered on the first day of an alternate-day schedule, with age, and with adiposity index. Growth hormone treatment of children with renal allografts who are growing poorly does not significantly affect glucose metabolism as assessed by oGTT.

Exon 7 Ncol restriction site within CYP21B (steroid 21-hydroxylase) is a normal polymorphism.

A point mutation within exon 7 producing an amino acid coding change and a recognition site for the endonuclease Ncol has been reported in the HLA-Bw47-linked CYP21A pseudogene and some mutant CYP21B (steroid 21-hydroxylase) genes of patients with congenital adrenal hyperplasia (CAH). Whether this mutation is deleterious was not demonstrated. We analyzed DNA from various subjects for the presence of the exon 7 Ncol site: group 1, 10 normal subjects; group 2, 11 patients with salt-losing CAH; and group 3, 18 members of an Amish pedigree in which 10 expressed HLA-Bw47 not linked to CAH. Southern blots of Ncol-digested genomic DNA which were hybridized with CYP21 cDNA showed that four subjects of group 1 had a heterozygous Ncol pattern. In group 2, seven patients had the Ncol site; two of them were homozygous for the site and had deletions of both CYP21B genes. The other five were heterozygous for the Ncol site, which was linked to a CYP21B deletion and a HLA-Bw47 haplotype. In group 3, no one exhibited the exon 7 Ncol site. To map the Ncol sites to CYP21A or CYP21B in the normal subjects, DNA from the four Ncol heterozygous subjects was double digested with Ncol and Mbol and hybridized with CYP21 cDNA. Ncol-Mbol fragments unique to CYP21A were identified in all four, but the smaller CYP21B-specific fragments were not detected. Their genomic DNA in the region of exon 7 (bases +1167 to +2058) was then amplified, cloned, and sequenced.(ABSTRACT TRUNCATED AT 250 WORDS)

Endocrine evaluation of adults with mild hypospadias.

The goal of our study was to determine the frequency of endocrine abnormalities in sexually mature men with mild hypospadias. For this purpose we evaluated 16 men 22 to 52 years old with mild hypospadias. All patients had a urethral meatus distal to the penoscrotal junction. Serum concentrations of luteinizing hormone, follicle-stimulating hormone and testosterone were measured in all subjects. Dihydrotestosterone and testosterone precursors also were determined in men who had abnormally elevated levels of luteinizing hormone. The level of serum luteinizing hormone in patients was significantly higher than that of normal men, whereas the levels of serum follicle-stimulating hormone and testosterone were similar to those of normal subjects. Of the 16 men with mild hypospadias 3 had abnormally elevated levels of gonadotropins: 2 had primary gonadal dysfunction (elevated luteinizing hormone and follicle-stimulating hormone with low or normal testosterone) and 1 had hormonal evidence of partial androgen insensitivity (elevated luteinizing hormone, normal follicle-stimulating hormone with high normal testosterone and dihydrotestosterone). These patients are rather unique, since they did not have other compromising factors to account for the gonadal failure. In summary, our data demonstrated the presence of endocrine dysfunction in men with mild hypospadias.

Two distinct areas of unequal crossingover within the steroid 21-hydroxylase genes produce absence of CYP21B.

We mapped crossover sites in chimeric, recombinant CYP21 genes from six patients with salt-losing congenital adrenal hyperplasia (CAH). Nucleotide sequences unique to the CYP21A pseudogene or to the active CYP21B gene were mapped using gene-specific restriction sites and oligonucleotide hybridizations. Each chimeric CYP21 gene in the CYP21-deletion linked haplotypes contained sequences near the 5' end that were characteristic of CYP21A and only a single transition from sequences of CYP21A to those of CYP21B at the 3' end. The transitions all occurred within either of two discrete regions (+470 to +999 and +1375 to +1993). All eight chimeric CYP21 genes coupled with HLA-Bw47 in five unrelated patients had the CYP21A-CYP21B sequence transition within the same gene region (+1375 to +1993). One of the three other "CYP21B deletion" haplotypes (HLA-B7) had a sequence transition within this same region, while in the other two haplotypes (HLA-B61 and HLA-B18) the transition occurred between base pairs +470 and +999. By contrast, both CYP21 genes in a haplotype containing a gene conversion of CYP21B to CYP21A contained apparent transitions between sequences of CYP21A and CYP21B. We conclude that a single, unequal crossingover between the CYP21A and the CYP21B genes yields deletion of the active CYP21 gene and salt-losing CAH and that these crossingovers do not occur randomly within the CYP21 genes of our patients.