Primus E. Mullis

Prevalence of Human GH-1 Gene Alterations in Patients with Isolated Growth Hormone Deficiency

Human GH is encoded by the GH-1 gene which belongs to the GH gene cluster encompassing a distance of about 65 kb on the long arm of chromosome 17. Familial isolated growth hormone deficiency (IGHD) is associated with at least four Mendelian disorders. These include two forms that have autosomal recessive inheritance (IGHD types IA and IB) as well as autosomal dominant(IGHD type II) and X-linked (IGHD III) forms. The aim of our study was to evaluate the prevalence of all GH-1 gene alterations by sequencing the whole GH-1 gene after PCR amplification among 151 affected subjects from 83 families with severe IGHD (height: <-4.5 SD score). A high frequency of GH-1 gene alterations was found in families with IGHD type IA (8/12, 66.7%), whereas only a low frequency of GH-1 gene defects was present in all the other GH-deficient families (7/71, 9.9%). The absolute frequency of GH-1 gene deletions was 8.7% (6/69), 11.8% (4/34), and 18.7% (9/48) in Northern Europeans, Mediterraneans, and Asians, respectively, giving an overall frequency of 12.5% (19/151). The sizes of the deletions were heterogeneous with the most frequent (78%) being 6.7 kb. In addition, 6% (9/151) of the patients presented GH-1 gene mutations such as frameshift, stop codon and splicing error. Furthermore, total GH-1 gene abnormalities varied among different populations from 11.6% in Northern Europe, 14.7% in Mediterranean countries and 31.2% in Asia. Most striking, however, was the low frequency rate of 1.7% (2/119) of GH-1 gene mutations responsible for the most common phenotype of IGHD, namely type IB, among the subjects characterized by the production of deficient but detectable amounts of GH after provocative stimuli. This finding underlines the necessity to focus rather on the promoter region of the GH-1 gene (cis-acting elements and trans-acting factors), and on other candidate genes specific for the GH axis than the GH-1 gene itself to define genetically the IGHD type IB phenotype in more detail.

Expanding the Spectrum of Mutations in GH1 and GHRHR: Genetic Screening in a Large Cohort of Patients with Congenital Isolated Growth Hormone Deficiency

CONTEXT It is estimated that 3-30% of cases with isolated GH deficiency (IGHD) have a genetic etiology, with a number of mutations being reported in GH1 and GHRHR. The aim of our study was to genetically characterize a cohort of patients with congenital IGHD and analyze their characteristics. PATIENTS AND METHODS A total of 224 patients (190 pedigrees) with IGHD and a eutopic posterior pituitary were screened for mutations in GH1 and GHRHR. To explore the possibility of an association of GH1 abnormalities with multiple pituitary hormone deficiencies, we have screened 62 patients with either multiple pituitary hormone deficiencies (42 pedigrees), or IGHD with an ectopic posterior pituitary (21 pedigrees). RESULTS Mutations in GH1 and GHRHR were identified in 41 patients from 21 pedigrees (11.1%), with a higher prevalence in familial cases (38.6%). These included previously described and novel mutations in GH1 (C182X, G120V, R178H, IVS3+4nt, a>t) and GHRHR (W273S, R94L, R162W). Autosomal dominant, type II IGHD was the commonest form (52.4%), followed by type IB (42.8%) and type IA (4.8%). Patients with type II IGHD had highly variable phenotypes. There was no difference in the endocrinology or magnetic resonance imaging appearance between patients with and without mutations, although those with mutations presented with more significant growth failure (height, -4.7 +/- 1.6 SDS vs. -3.4 +/- 1.7 SDS) (P = 0.001). There was no apparent difference between patients with mutations in GH1 and GHRHR. CONCLUSIONS IGHD patients with severe growth failure and a positive family history should be screened for genetic mutations; the evolving endocrinopathy observed in some of these patients suggests the need for long-term follow-up.

A cross‐sectional international survey of continuous subcutaneous insulin infusion in 377 children and adolescents with type 1 diabetes mellitus from 10 countries

Objective:  To document current practices using continuous subcutaneous insulin infusion (CSII) by downloading electronically the 90‐d pump data held within the pump memory and relating that to clinical data from children and adolescents in different pediatric diabetes centers from Europe and Israel.

Ten Novel Mutations in the NR5A1 Gene Cause Disordered Sex Development in 46,XY and Ovarian Insufficiency in 46,XX Individuals

CONTEXT Steroidogenic factor-1 (SF-1/NR5A1) is a nuclear receptor that regulates adrenal and reproductive development and function. NR5A1 mutations have been detected in 46,XY individuals with disorders of sexual development (DSD) but apparently normal adrenal function and in 46,XX women with normal sexual development yet primary ovarian insufficiency (POI). OBJECTIVE A group of 100 46,XY DSD and two POI was studied for NR5A1 mutations and their impact. DESIGN Clinical, biochemical, histological, genetic, and functional characteristics of the patients with NR5A1 mutations are reported. SETTING Patients were referred from different centers in Spain, Switzerland, and Turkey. Histological and genetic studies were performed in Barcelona, Spain. In vitro studies were performed in Bern, Switzerland. PATIENTS A total of 65 Spanish and 35 Turkish patients with 46,XY DSD and two Swiss 46,XX patients with POI were investigated. MAIN OUTCOME Ten novel heterozygote NR5A1 mutations were detected and characterized (five missense, one nonsense, three frameshift mutations, and one duplication). RESULTS The novel NR5A1 mutations were tested in vitro by promoter transactivation assays showing grossly reduced activity for mutations in the DNA binding domain and variably reduced activity for other mutations. Dominant negative effect of the mutations was excluded. We found high variability and thus no apparent genotype-structure-function-phenotype correlation. Histological studies of testes revealed vacuolization of Leydig cells due to fat accumulation. CONCLUSIONS SF-1/NR5A1 mutations are frequently found in 46,XY DSD individuals (9%) and manifest with a broad phenotype. Testes histology is characteristic for fat accumulation and degeneration over time, similar to findings observed in patients with lipoid congenital adrenal hyperplasia (due to StAR mutations). Genotype-structure-function-phenotype correlation remains elusive.

Pioglitazone inhibits androgen production in NCI-H295R cells by regulating gene expression of CYP17 and HSD3B2.

Thiazolidinediones (TZDs) such as pioglitazone and rosiglitazone are widely used as insulin sensitizers in the treatment of type 2 diabetes. In diabetic women with polycystic ovary syndrome, treatment with pioglitazone or rosiglitazone improves insulin resistance and hyperandrogenism, but the mechanism by which TZDs down-regulate androgen production is unknown. Androgens are synthesized in the human gonads as well as the adrenals. We studied the regulation of androgen production by analyzing the effect of pioglitazone and rosiglitazone on steroidogenesis in human adrenal NCI-H295R cells, an established in vitro model of steroidogenesis of the human adrenal cortex. Both TZDs changed the steroid profile of the NCI-H295R cells and inhibited the activities of P450c17 and 3βHSDII, key enzymes of androgen biosynthesis. Pioglitazone but not rosiglitazone inhibited the expression of the CYP17 and HSD3B2 genes. Likewise, pioglitazone repressed basal and 8-bromo-cAMP-stimulated activities of CYP17 and HSD3B2 promoter reporters in NCI-H295R cells. However, pioglitazone did not change the activity of a cAMP-responsive luciferase reporter, indicating that it does not influence cAMP/protein kinase A/cAMP response element-binding protein pathway signaling. Although peroxisome proliferator-activated receptor γ (PPARγ) is the nuclear receptor for TZDs, suppression of PPARγ by small interfering RNA technique did not alter the inhibitory effect of pioglitazone on CYP17 and HSD3B2 expression, suggesting that the action of pioglitazone is independent of PPARγ. On the other hand, treatment of NCI-H295R cells with mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibitor 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) enhanced promoter activity and expression of CYP17. This effect was reversed by pioglitazone treatment, indicating that the MEK/ERK signaling pathway plays a role in regulating androgen biosynthesis by pioglitazone.

Genetic evaluation of short stature

After a proper medical history, growth analysis and physical examination of a short child, followed by radiological and laboratory screening, the clinician may decide to perform genetic testing. We propose several clinical algorithms that can be used to establish the diagnosis. GH1 and GHRHR should be tested in children with severe isolated growth hormone deficiency and a positive family history. A multiple pituitary dysfunction can be caused by defects in several genes, of which PROP1 and POU1F1 are most common. GH resistance can be caused by genetic defects in GHR, STAT5B, IGF1, IGFALS, which all have their specific clinical and biochemical characteristics. IGF-I resistance is seen in heterozygous defects of the IGF1R. If besides short stature additional abnormalities are present, these should be matched with known dysmorphic syndromes. If no obvious candidate gene can be determined, a whole genome approach can be taken to check for deletions, duplications and/or uniparental disomies.

The Aromatase Cytochrome P-450 and Its Clinical Impact

Cytochrome P-450 aromatase (P450arom), the key enzyme for estrogen biosynthesis, is encoded by a single gene, namely the CYP19 gene, localized on 15q21.2. The human CYP19 gene spans about 123 kb with a coding region of 9 exons (about 30 kb, exon II–exon X). Although there are a number of alternative first exons and nine different transcriptional start sides with individual promoters that permit tissue-specific regulation of expression, the protein expressed in these various tissue sites (placenta, adipose tissue, brain, bone, ovary, etc.) is the same regardless of the promoter used. P450arom catalyzes the conversion of testosterone to estradiol, of androstenedione to estrone, and of 16α-hydroxylated dehydroepiandrosterone to estriol. As not only androgens but also estrogens are of importance, particularly in the male pubertal development, including bone changes which were classically considered mostly androgen dependent, the features of the aromatase deficiency syndrome in affected boys and girls as well as adult males and females are discussed. There is growing awareness that androgens and estrogens have general metabolic roles that reach far beyond reproductive processes. For instance, estrogen has a significant impact on carbohydrate and lipid metabolism, vascular function, and arteriosclerosis. In addition, extragonadal estrogen biosynthesis plays an important but often underestimated physiological and pathophysiological role, for example in breast cancer and endometriosis. Based on that knowledge, progress has been made as far as treatment and follow-up of these disorders are concerned. In addition, there is a focus on the treatment of children suffering from a lack of P450arom activity.

The FGFRL1 receptor is shed from cell membranes, binds fibroblast growth factors (FGFs), and antagonizes FGF signaling in Xenopus embryos.

FGFRL1 (fibroblast growth factor receptor like 1) is the fifth and most recently discovered member of the fibroblast growth factor receptor (FGFR) family. With up to 50% amino acid similarity, its extracellular domain closely resembles that of the four conventional FGFRs. Its intracellular domain, however, lacks the split tyrosine kinase domain needed for FGF-mediated signal transduction. During embryogenesis of the mouse, FGFRL1 is essential for the development of parts of the skeleton, the diaphragm muscle, the heart, and the metanephric kidney. Since its discovery, it has been hypothesized that FGFRL1 might act as a decoy receptor for FGF ligands. Here we present several lines of evidence that support this notion. We demonstrate that the FGFRL1 ectodomain is shed from the cell membrane of differentiating C2C12 myoblasts and from HEK293 cells by an as yet unidentified protease, which cuts the receptor in the membrane-proximal region. As determined by ligand dot blot analysis, cell-based binding assays, and surface plasmon resonance analysis, the soluble FGFRL1 ectodomain as well as the membrane-bound receptor are capable of binding to some FGF ligands with high affinity, including FGF2, FGF3, FGF4, FGF8, FGF10, and FGF22. We furthermore show that ectopic expression of FGFRL1 in Xenopus embryos antagonizes FGFR signaling during early development. Taken together, our data provide strong evidence that FGFRL1 is indeed a decoy receptor for FGFs.

Role of DNA methylation in the tissue-specific expression of the CYP17A1 gene for steroidogenesis in rodents

The CYP17A1 gene is the qualitative regulator of steroidogenesis. Depending on the presence or absence of CYP17 activities mineralocorticoids, glucocorticoids or adrenal androgens are produced. The expression of the CYP17A1 gene is tissue as well as species-specific. In contrast to humans, adrenals of rodents do not express the CYP17A1 gene and have therefore no P450c17 enzyme for cortisol production, but produce corticosterone. DNA methylation is involved in the tissue-specific silencing of the CYP17A1 gene in human placental JEG-3 cells. We investigated the role of DNA methylation for the tissue-specific expression of the CYP17A1 gene in rodents. Rats treated with the methyltransferase inhibitor 5-aza-deoxycytidine excreted the cortisol metabolite tetrahydrocortisol in their urine suggesting that treatment induced CYP17 expression and 17alpha-hydroxylase activity through demethylation. Accordingly, bisulfite modification experiments identified a methylated CpG island in the CYP17 promoter in DNA extracted from rat adrenals but not from testes. Both methyltransferase and histone deacetylase inhibitors induced the expression of the CYP17A1 gene in mouse adrenocortical Y1 cells which normally do not express CYP17, indicating that the expression of the mouse CYP17A1 gene is epigenetically controlled. The role of DNA methylation for CYP17 expression was further underlined by the finding that a reporter construct driven by the mouse -1041 bp CYP17 promoter was active in Y1 cells, thus excluding the lack of essential transcription factors for CYP17 expression in these adrenal cells.

Reduction in hepatic drug metabolizing CYP3A4 activities caused by P450 oxidoreductase mutations identified in patients with disordered steroid metabolism

Cytochrome P450 3A4 (CYP3A4), the major P450 present in human liver metabolizes approximately half the drugs in clinical use and requires electrons supplied from NADPH through NADPH-P450 reductase (POR, CPR). Mutations in human POR cause a rare form of congenital adrenal hyperplasia from diminished activities of steroid metabolizing P450s. In this study we examined the effect of mutations in POR on CYP3A4 activity. We used purified preparations of wild type and mutant human POR and in vitro reconstitution with purified CYP3A4 to perform kinetic studies. We are reporting that mutations in POR identified in patients with disordered steroidogenesis/Antley-Bixler syndrome (ABS) may reduce CYP3A4 activity, potentially affecting drug metabolism in individuals carrying mutant POR alleles. POR mutants Y181D, A457H, Y459H, V492E and R616X had more than 99% loss of CYP3A4 activity, while POR mutations A287P, C569Y and V608F lost 60-85% activity. Loss of CYP3A4 activity may result in increased risk of drug toxicities and adverse drug reactions in patients with POR mutations.