Developmental Biology of GH Secretion, Growth and Treatment

P. -E. Mullis

• Publisher: S. Karger
• Year: 2012
• ISBN:
  9783318022452
• Topic: Medicine
• Subtopic: Endocrinology & Metabolism

In the last few years, rapid progress has taken place in our understanding of the developmental biology of GH secretion and the pivotal role it plays in growth.This book keeps the reader updated on the most important developmental aspects and influences leading to changes in terms of clinical views. In ten chapters, well-known scientists and clinicians cover some of the most important progress made in recent times. The first chapters discuss pituitary gland development and imaging in detail followed by a comprehensive presentation of the genetics of the GH axis. Further chapters present a detailed overview of the epigenetics and bioinformatics of GH.This collection of up-to-date investigative data and reviews is of relevance not only to scientists involved in endocrinology but also to any physician interested in growth and development.

Mullis P-E (ed): Developmental Biology of GH Secretion, Growth and Treatment.
Endocr Dev. Basel, Karger, 2012, vol 23, pp 71–85 (DOI: 10.1159/000341755)

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Bioinformatics Tools and Databases for the Study of Human Growth Hormone

Amit V. Pandey

Pediatric Endocrinology, Diabetology and Metabolism, Department of Clinical Research, University of Bern, and University Children's Hospital Bern, Bern, Switzerland

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Abstract

Advances in novel molecular biological diagnostic methods are changing the way of diagnosis and study of metabolic disorders like growth hormone deficiency. Faster sequencing and genotyping methods require strong bioinformatics tools to make sense of the vast amount of data generated by modern laboratories. Advances in genome sequencing and computational power to analyze the whole genome sequences will guide the diagnostics of future. In this chapter, an overview of some basic bioinformatics resources that are needed to study metabolic disorders are reviewed and some examples of bioinformatics analysis of human growth hormone gene, protein and structure are provided.

Copyright © 2012 S. Karger AG, Basel

Advances in molecular genetics and bioinformatics are changing the ways of detection and treatment of genetic diseases. Novel DNA sequencing and mapping tools together with the availability of the human genome have made it possible to identify the molecular basis of complex genetic disorders. Study of human growth hormone (GH) has been at the forefront of advances in modern diagnostic and treatment methods. In the 21st century, endocrinology has evolved from the measurement of hormones and metabolites to genetic analysis of whole pathways involved in hormone synthesis and action, including biosynthesis of hormones, genes and proteins involved in hormone synthesis, interaction of hormones with their receptors as well as regulation of genes involved in hormone biosynthesis and action pathways in a cellular and tissue-specific manner. The importance of human GH in clinical medicine has been recognized for a long time. Human GH was one of the first genes to be studied at the molecular genetic level and pioneered the molecular biological studies for cloning in bacteria and production as recombinant molecules [1, 2]. Human GH was also among the first recombinant proteins that was made for therapeutic use and contributed to the establishment of the pioneering biotechnology company Genentech in San Francisco, Calif., USA.

Molecular biological methods allow detection of rare genetic disorders when biochemical laboratory measurements are not clear as well as in pediatric and neonatal diagnosis where sample size is often limited. The latest advancements in the field have been the availability of the human genome sequence and novel sequencing technologies. An almost complete catalogue of human genes has allowed endocrinologists to identify candidate genes for many genetic disorders and subsequent sequencing of genetic material from patients had led to identification of the molecular basis of many hormonal and metabolic defects.

Basic Analytical Methods

Identification of the exact location of mutations is of importance to clinical laboratories as a wide variation in severity of disease could be seen in disorders related to GH, often due to the wide nature of point mutations in the GH1 gene with each mutation resulting in a specific effect. Functional genomics characterization is performed by identification of the exact location of mutations followed by the biochemical characterization of recombinant genes/proteins that replicate the patient DNA sequences in an experimental setup. Modern diagnostic techniques employ DNA amplification-based methods and require only a small amount of DNA for most diagnostic methods.

PCR and Sequencing

It is advisable to use primer design software tools available at bioinformatics resources rather than relying on manual design. Primer3Plus provides a web interface to the popular Primer3 program with a task oriented approach. Several different options for primer design are available including sequencing and cloning as well as design of probes for Northern blot analysis, etc. (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi). Primers that display ‘significant’ homology to alternative targets should be discarded. The primer blast tool available at NCBI can screen for mismatches and alternative binding sites (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). DNA sequencing is still the most reliable method to confirm a genetic defect and provide the exact location and nature of the disorder. Sequencing of DNA is generally no longer performed in individual laboratories and has been relegated to central facilities or outsourced to private companies providing sequencing services. The automated and efficient sequencing technology has allowed comparison of normal and patient DNA sequences without manually reading the chromatograms. One efficient tool for automated mutation analysis from DNA sequence traces is Mutation Surveyor software from Softgenetics (http://www.softgenetics.com/mutationSurveyor.html). Mutation Surveyor is available for academic labs with simultaneous reading of 48 samples from Sanger Sequencing traces provided by automated dye terminator sequencers. The Mutation Surveyor can locate genetic variants, SNPs and Indel mutations between reference traces and sample/patient traces and has a several reporting possibilities for both research and laboratory diagnostics.

Diagnostics of the Future: SNP/Mutation Genotyping and Next Generation Sequencing

The high-density oligonucleotide SNP arrays are formed by spotting a large number of probes on small chips, allowing multiple SNPs to be probed simultaneously [3]. Oligonucleotide microarray methods currently have lower specificity and sensitivity compared to sequencing but the amount of SNPs that can be checked in a single analysis is of great advantage. Advances in a new generation of SNP chips that can include known disease-causing mutations will make it possible to screen for variations in GH1 and related genes in a single chip based assay. In the longer term, a whole genome sequencing approach may replace current single gene detection approaches. In the past few years, the cost of genome sequencing has come down considerably. Once the costs drop below USD 1,000 per genome and reliability can be of clinical diagnostic standards, it would be possible to order whole genome sequencing of newborns rather than multiple individual genetic tests. Such an approach would likely be cost effective when multiple blood drawings, individual sequencing and analysis that is currently used is replaced by a single genome sequencing test. This will allow analysis of not only GH1 but all related genes and pathways, promoters, inducers, transcription factors etc. that are likely to have an impact on GH1 based effects. Moreover, once the whole genome sequence of a patient is available, different queries can be performed to check for any individual gene that will allow clinicians to get the answers in a very fast manner compared to currently available methods.

Functional Analysis with Recombinant Proteins and Cell-Based Assays

Once a defect has been identified, the GH gene from patients can be cloned directly, or often the mutation/disordered can be replicated by changing the normal gene that is already available in laboratories in the form of standard DNA libraries. It is then possible to deduce the amino acid sequence of proteins and predict its structural and biophysical properties. Recombinant genes with ar...

Table of contents

• Cover Page
• Front Matter
• Pituitary Gland Development: An Update
• Pituitary Gland Imaging and Outcome
• Spectrum of Insulin-Like Growth Factor Deficiency
• Downstream Insulin-Like Growth Factor
• New Detection Methods of Growth Hormone and Growth Factors
• Imprinted Anomalies in Fetal and Childhood Growth Disorders: The Model of Russell-Silver and Beckwith-Wiedemann Syndromes
• Bioinformatics Tools and Databases for the Study of Human Growth Hormone
• Growth Hormone and Cell Growth
• From Endoplasmic Reticulum to Secretory Granules: Role of Zinc in the Secretory Pathway of Growth Hormone
• Isolated Growth Hormone Deficiency Type 2: From Gene to Therapy
• Author Index
• Subject Index