Jürgen Klammt

Pituitary transcription factors in the aetiology of combined pituitary hormone deficiency

The somatotropic axis is the central postnatal regulator of longitudinal growth. One of its major components--growth hormone--is produced by the anterior lobe of the pituitary, which also expresses and secretes five additional hormones (prolactin, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, adrenocorticotropic hormone). Proper development of the pituitary assures the regulation of critical processes such as metabolic control, puberty and reproduction, stress response and lactation. Ontogeny of the adenohypophysis is orchestrated by inputs from neighbouring tissues, cellular signalling molecules and transcription factors. Perturbation of expression or function of these factors has been implicated in the aetiology of combined pituitary hormone deficiency (CPHD). Mutations within the genes encoding for the transcription factors LHX3, LHX4, PROP1, and POU1F1 (PIT1) that act at different stages of pituitary development result in unique patterns of hormonal deficiencies reflecting their differential expression during organogenesis. In the case of LHX3 and LHX4 the phenotype may include extra-pituitary manifestations due to the function of these genes/proteins outside the pituitary gland. The remarkable variability in the clinical presentation of affected patients indicates the influence of the genetic background, environmental factors and possibly stochastic events. However, in the majority of CPHD cases the aetiology of this heterogeneous disease remains unexplained, which further suggests the involvement of additional genes. Identification of these factors might also help to close the gaps in our understanding of pituitary development, maintenance and function.

IGF signaling defects as causes of growth failure and IUGR

A substantial portion of children born small for gestational age (SGA) fail to catch up height, despite normal or even elevated insulin-like growth factor (IGF1) serum levels. In most cases, the etiology of the apparent IGF1 resistance is regarded as idiopathic. However, the recent identification of human IGF1 and IGF1 receptor (IGF1R) mutations, as well as information obtained from transgenic animals, points to a strong genetic component being of pivotal importance in the development of growth retardation. These findings direct attention to molecules downstream of the IGF1R, which have both growth-promoting and, to a lesser extent, metabolic functions. Therefore, defects in these molecules are likely to participate in the etiology of human SGA.

A Role for Leptin in Sexual Maturation and Puberty

Leptin, the ob gene product, is involved in the regulation of body weight in rodents, primates and humans. It provides a molecular basis for the lipostatic theory of the regulation of energy balance. White adipose tissue and placenta are the main sites of leptin synthesis. There is also evidence of ob gene expression in brown fat. Leptin seems to play a key role in the control of body fat stores by coordinated regulation of feeding behaviour, metabolic rate, autonomic nervous system regulation and body energy balance. Apart from the function of leptin in the central nervous system on the regulation of energy balance, it may well be one of the hormonal factors that signal to the brain the body’s readiness for sexual maturation and reproduction. During late pregnancy and at birth when maternal fat stores have been developed, leptin levels are high. During these developmental stages leptin could be a messenger molecule signalling the adequacy of the fat stores for reproduction and maintenance of pregnancy. At later stages of gestation leptin could signal the expansion of fat stores in order to prepare the expectant mother for the energy requirements of full-term gestation, labour and lactation. Leptin serum concentrations change during pubertal development in rodents, primates and humans. In girls, leptin serum concentrations increase dramatically as pubertal development proceeds. The pubertal rise in leptin levels parallels the increase in body fat mass. In contrast, leptin levels increase shortly before and during the early stages of puberty in boys and decline thereafter. Testosterone has been found to suppress leptin synthesis by adipocytes both in vivo and in vitro. The decline of leptin levels in late puberty in boys accompanies increased androgen production during that time and most likely reflects suppression of leptin by testosterone and a decrease in fat mass and relative increase in muscle mass during late puberty in males. This overview focuses on those topics of leptin research which are of particular interest in reproductive and adolescent medicine.

IGF1R mutations as cause of SGA.

Until 2003 monogenetic aberrations that lead to a child that is born too small for gestational age (SGA) were poorly defined. With the first report of mutations within the insulin-like growth factor type 1 receptor (IGF1R) gene in two non-syndromic patients born SGA, who failed to thrive despite normal or even elevated IGF1 serum concentrations the concept of IGF1 resistance has been established. The identification of additional individuals bearing IGF1R mutations along with comparative, genetic, structural and biochemical studies has provided evidence for the pathogenic impact of the IGF1R mutations on human longitudinal growth. However, the variability in the occurrence of additional clinical manifestations, such as developmental delay, might indicate that the pleiotropic functions of the IGF-IGF1R system are partially redundant. It is apparent that we have just begun to unravel the multifaceted IGF1R actions at the interface of growth control, maintenance of metabolic homeostasis and neurodevelopment and neural protection.

Body fat mass, leptin and puberty.

Leptin, the ob gene product, provides a molecular basis for the lipostatic theory of the regulation of energy balance. Leptin circulates as a monomeric 16 kDa protein in rodent and human plasma and is also bound to leptin binding proteins that may form large high molecular weight complexes. Initial models of leptin action included leptin-deficient ob/ob mice and leptin-insensitive db/db mice. Peripheral or central administration of leptin reduced body weight, adiposity, and food intake in ob/ob mice but not in db/db mice. In ob/ob mice leptin treatment restored fertility. Leptin interacts with many messenger molecules in the brain. For example, leptin suppresses neuropeptide Y (NPY) expression in the arcuate nucleus. Increased NPY activity has an inhibitory effect on the gonadotropin axis and represents a direct mechanism for inhibiting sexual maturation and reproductive function in conditions of food restriction and/or energy expenditure. By modulating the hypothalamo-pituitary-gonadal axis both directly and indirectly, leptin may thus serve as the signal from fat to the brain about the adequacy of fat stores for pubertal development and reproduction. Normal leptin secretion is necessary for normal reproductive function to proceed and leptin may be a signal allowing for the point of initiation of and progression toward puberty.

Homozygous mutation of the IGF1 receptor gene in a patient with severe pre- and postnatal growth failure and congenital malformations

BACKGROUND Heterozygous mutations in the IGF1 receptor (IGF1R) gene lead to partial resistance to IGF1 and contribute to intrauterine growth retardation (IUGR) with postnatal growth failure. To date, homozygous mutations of this receptor have not been described. SUBJECT A 13.5-year-old girl born from healthy first-cousin parents presented with severe IUGR and persistent short stature. Mild intellectual impairment, dysmorphic features, acanthosis nigricans, and cardiac malformations were also present. METHODS Auxological and endocrinological profiles were measured. All coding regions of the IGF1R gene including intron boundaries were amplified and directly sequenced. Functional characterization was performed by immunoblotting using patients fibroblasts. RESULTS IGF1 level was elevated at 950NG/ML (+7 S.D.). Fasting glucose level was normal associated with high insulin levels at baseline and during an oral glucose tolerance test. Fasting triglyceride levels were elevated. sequencing of the IGF1R gene led to the identification of a homozygous variation in exon 2: c.119G>T (p.Arg10Leu). As a consequence, IGF1-dependent receptor autophosphorylation and downstream signaling were reduced in patients fibroblasts. Both parents were heterozygous for the mutation. CONCLUSION The homozygous mutation of the IGF1R is associated with severe IUGR, dysmorphic features, and insulin resistance, while both parents were asymptomatic heterozygous carriers of the same mutation.

Identification of three novel plasminogen (PLG) gene mutations in a series of 23 patients with low PLG activity

Inherited severe hypoplasminogenaemia is a multisystemic disorder leading to deficient extravascular fibrinolysis. As a clinical consequence wound healing capacity of mucous membranes is markedly impaired leading to ligneous conjunctivitis and several other manifestations. Here we report the molecular genetic and clinical findings on 23 new cases with severe hypoplasminogenaemia. Homozygous or compound-heterozygous mutations in the plasminogen (PLG) gene were found in 16 of 23 patients (70%), three of which were novel mutations reported here for the first time (C166Y, Y264S, IVS10-7T/G). Compared to 79 previously published cases, clinical manifestations of the current group of patients showed higher percentages of ligneous periodontitis, congenital hydrocephalus, and involvement of the female genital tract. In contrast, involvement of the gastrointestinal or urogenital tract was not observed in any of the cases. Patients originated to a large extent (61%) from Turkey and the Middle East, and showed a comparably frequent occurrence of consanguinity of affected families and a greater female to male ratio than was derived from previous reports in the literature. Individual treatment of ligneous conjunctivitis included topical plasminogen or heparin eye drops, topical or systemic fresh frozen plasma, and surgical removal of ligneous pseudomembranes, mostly with modest or transient efficacy. In conclusion, the present study underscores the broad range of clinical manifestations in PLG-deficient patients with a trend to regional differences. Transmission of genetic and clinical data to the recently established Plasminogen Deficiency Registry should help to determine the prevalence of the disease and to develop more efficient treatment strategies.

Functional Characterization of a Heterozygous GLI2 Missense Mutation in Patients With Multiple Pituitary Hormone Deficiency

CONTEXT The GLI2 transcription factor is a major effector protein of the sonic hedgehog pathway and suggested to play a key role in pituitary development. Genomic GLI2 aberrations that mainly result in truncated proteins have been reported to cause holoprosencephaly or holoprosencephaly-like features, sometimes associated with hypopituitarism. OBJECTIVE Our objective was to determine the frequency of GLI2 mutations in patients with multiple pituitary hormone deficiency (MPHD). DESIGN Patients were selected from participants in the Genetics and Neuroendocrinology of Short Stature International Study (GeNeSIS) program. Patients with mutations within established candidate genes were excluded. PATIENTS A total of 165 patients with MPHD defined as GH deficiency and at least 1 additional pituitary hormone deficiency were studied regardless of the presence of extrapituitary clinical manifestations. MAIN OUTCOME MEASURES Prevalence of GLI2 variations in MPHD patients was assessed and detailed phenotypic characterization is given. Transcriptional activity of identified GLI2 variants was evaluated by functional reporter assays. RESULTS In 5 subjects, 4 heterozygous missense variants were identified, of which 2 are unpublished so far. One variant, p.R516P, results in vitro in a complete loss of protein function. In addition to GH deficiency, the carrier of the mutation demonstrates deficiency of thyrotrope and gonadotrope function, a maldescended posterior pituitary lobe, and polydactyly, but no midline defects. CONCLUSIONS For the first time, we show that heterozygous amino acid substitutions within GLI2 may lead to MPHD with mild extrapituitary findings. The phenotype of GLI2 mutations is variable, and penetrance is incomplete. GLI2 mutations are associated with anterior pituitary hypoplasia, and frequently, ectopy of the posterior lobe occurs.

Glucose regulates expression of the nerve growth factor (NGF) receptors TrkA and p75NTR in rat islets and INS-1E β-cells

BACKGROUND The function and survival of pancreatic beta-cells strongly depend on glucose concentration and on autocrine secretion of peptide growth factors. NGF and its specific receptors TrkA and p75NTR play a pivotal role in islet survival and glucose-dependent insulin secretion. We therefore investigated whether or not glucose concentration influences expression of TrkA and p75NTR in rat islets and in INS-1E beta-cells at the mRNA and protein level (INS-1E). METHODS Gene expression of the NGF receptors TrkA and p75NTR but also of the metabolic gene liver-type pyruvate kinase (L-PK) and the neurotrophin receptors TrkB and TrkC was studied by semi-quantitative PCR and by real-time PCR in islets and INS-1E beta-cells. RESULTS In rat islets, high glucose exposure (25 mmol/l) increased gene expression of TrkA, p75NTR and L-PK. Expression of TrkA, p75NTR and L-PK reflected insulin secretion at the respective glucose concentration. In rat INS-1E insulinoma cells, expression of L-PK and p75NTR was suppressed by low glucose as in the islets, while expression of TrkA was strongly increased by low glucose levels and thus was regulated differently than in islets. Expression of TrkB and TrkC was not regulated by glucose concentration at all. TrkA protein was regulated in the same fashion as its mRNA expression, while p75NTR protein was not significantly regulated within 24 h. CONCLUSION Glucose interacts with gene expression of TrkA and p75NTR that are strongly involved in beta-cell growth and glucose-dependent insulin secretion. The fact that TrkA expression is regulated the opposite way in islets and in INS-1E beta-cells might reflect their specific grade of differentiation and tendency to proliferate.

Clinical Examples of Disturbed IGF Signaling: Intrauterine and Postnatal Growth Retardation due to Mutations of the Insulin-Like Growth Factor I Receptor (IGF-IR) Gene

The insulin-like growth factor-I receptor (IGF-IR) is an important mediator of cell proliferation and longitudinal bone growth. The IGF-IR is closely related to the insulin receptor with a homology of 80–95% in the tyrosine kinase domain [1,2]. IGF-I binding to the IGF-IR causes the transmembrane activation of the tyrosine kinase activity of the IGF-IR [4]. In contrast to the insulin receptor, an impairment of the closely related IGF-IR has only been suggested on rare occasions under clinical circumstances: for instance, monoallelic loss of chromosome 15q, mutations of the IGF-I receptor gene and loss of one copy of the IGF-I receptor gene again due to deletions of the distal long arm of chromosome 15 have been found in patients with intrauterine growth retardation and postnatal growth deficit [5]. Binding of IGF-I to erythrocytes in short children with IUGR has been shown to be lower than in children with normal height [5]. The number of IGF-I receptor copies on human fibroblasts seems to be predictive of their proliferative response to IGF-I. Hemizygosity for IGFIR can cause primary IGF-I resistance despite normal or even elevated GH and/or IGF-I serum concentrations. IGF-IR gene knockout experiments in mice have shown that mice carrying null mutations of both alleles exhibit severe and those of one allele moderate embryonic and postnatal growth deficiency [7,8,9]. Recently, a compound heterozygous mutation of the human IGF-IR gene and a nonsense mutation (Arg59stop) that reduced the number of IGF-I receptors on fibroblasts from the affected child have been described in children with intrauterine growth retardation and highly elevated IGF-I levels [10]. The phenotype of patients with IGF-I receptor mutations and/or reduction of IGF-I receptor numbers known to date are being presented. Their relevance for clinical medicine and a hypothesized potential for future research on growth and growth disorders is being discussed.