The genetic toolbox: dissecting pathways to growth

The role of growth hormone (GH) has been established for many decades, but it is only with the advent of advanced molecular genetic techniques that researchers have started to piece together the precise pathways leading to growth.

Here we review seven key papers, chosen by Professor Andrew Dauber, from Cincinnati Children’s Hospital Medical Center in Ohio, USA, which document the pivotal findings that shed light on the process of signalling through the GH/insulin-like growth factor (IGF)-I axis.

The research largely represents the extreme end of the growth disorder spectrum, with a single mutation having a profound effect on a patient’s height, but Dauber stresses that “they teach us a lot about the underlying physiology and the role that those genes play in normal biology.”

Laron dwarfism was first described in 1966, as an autosomal recessive syndrome with many clinical similarities to isolated GH deficiency. The characteristic low levels of IGF-I despite increased levels of GH implied a role for the GH receptor, but it was not until 1989 that researchers provided direct evidence of this hypothesis. Michel Goossens (CHU Henri Mondor, Créteil, France) and colleagues identified a likely culprit mutation in the GH receptor gene of a family with consanguineous parents with normal phenotypes who had four children, all affected by Laron dwarfism.1

All of the children were homozygous for a single base-pair substitution resulting in serine being present at a position in the extracellular domain of the GH receptor where phenylalanine is invariably found across multiple species. This, combined with the lack of GH-binding activity in the study patients, led the team to conclude that the mutation was probably causative, providing the first direct evidence for the role of the GH receptor in growth.

In contrast to this study, which actively sought the genetic basis for a condition, another two landmark reports detail more serendipitous findings; direct evidence for the involvement of STAT5 and the IGF-I receptor in growth signalling both emerged from researchers’ efforts to identify the cause of short stature in an individual child, while the surprising findings of another case study challenged the importance of the acid-labile subunit (ALS) in GH/IGF-I signalling.

In one case, Ron Rosenfeld (Oregon Health and Science University, Portland, USA) and co-researchers studied a girl whose height was 7.5 standard deviations (SD) below the average for her age, of 16.5 years.2 She had elevated GH levels and low IGF-I levels, consistent with GH resistance, yet her GH receptor gene had no mutations.

Administration of GH to the patient’s fibroblasts failed to produce the expected increase in IGF-I, leading the researchers to focus on the signalling pathways between these two molecules. This revealed a homozygous missense mutation in STAT5b, which rendered the protein incapable of activation by GH, despite being stably expressed.

Activated STAT5b in turn starts a signalling cascade that leads to increased transcription of IGF-I, IGF binding protein 3 and ALS. Together these molecules form a ternary complex, in which ALS stabilises the binding of IGF-I to IGF binding protein 3 and extends the half-life of circulating IGF-I. As the majority (80–85%) of IGF-I in the body is in this complex form, it was thought to be the main pathway through which GH exerts its effects.3

However, the case of a boy who entirely lacked ALS challenged this notion. The patient had a frameshift point mutation, which prevented production of ALS; he therefore had no ternary complexes and his plasma level of IGF-I was more than 5 SD below the average.

Yet, although the patient was referred to Horacio Domené (Ricardo Gutiérrez Children’s Hospital, Buenos Aires, Argentina) and colleagues partly for growth restriction, this was less severe than might be expected, at 2.05 SD below average. The team speculated that growth in their patient had been sustained by free or locally produced IGF-I, implying that total circulating IGF-I levels are less critical than previously believed.

A third case study supplied direct evidence that IGF-I is crucial not just for postnatal growth but also for intrauterine growth. The report featured a 15.8-year-old boy who had a birth length 5.4 SD below average and continued to have severe growth retardation throughout childhood, so that, by the time of the report, his height was 6.9 SD below average.4

This patient had elevated GH levels and undetectable serum IGF-I, and Adrian Clark and colleagues, from St Bartholomew’s Hospital in London, UK, found that this was caused by a mutation of the IGF-I gene itself. Their patient was missing exons 4 and 5, resulting in a truncated IGF-I protein.

Evidence for the role of the IGF-I receptor in pre- and postnatal growth came from Steven Chernausek (Cincinnati Children’s Hospital Medical Center, Ohio, USA) and co-workers, who sequenced the gene in 51 children with heights more than 2 SD below average.5 They identified two children, who both had intrauterine and postnatal growth restriction and both had mutations in the IGF-I receptor gene, one resulting in reduced IGF-I receptor function and the other in a reduced number of IGF-I receptors on the fibroblasts.

Although closely related to IGF-I, and well-recognised as a major influence on intrauterine growth, IGF-II has been considered less important for postnatal growth. However, a very recent paper detailed how a paternally inherited IGF-II nonsense mutation resulted in Silver–Russell syndrome.6

Thomas Eggermann (University Hospital, Aachen, Germany) and co-researchers found their patients lacked mutations known to cause Silver–Russell syndrome, but detected the IGF-II mutation using exome and Sanger sequencing. Two of the three affected children were very small at birth but responded to later growth hormone treatment and achieved an adult height close to the third centile; the third did not respond and had continued poor growth.

Another aspect of these case studies was the finding of other, non-growth symptoms that the researchers attributed to the mutations in the GH/IGF-I axis, implying pleiotropic actions of this pathway. For example, the boy with truncated IGF-I had, among other symptoms, profound sensorineural deafness and mental retardation, the girl with mutated STAT5b had a compromised immune response, and the Silver–Russell patients had symptoms including severe postnatal feeding problems, delayed development, mental retardation and hypotonia.

Postnatal growth is not regulated only by the GH/IGF-I axis. A notable example outside of this pathway is the SHOX (short stature homeobox-containing) gene, a transcription factor that is expressed in the growth plate.

SHOX was first described in 1997, by Gudrun Rappold (Heidelberg University, Germany) and colleagues.7 The cause of short stature in girls with Turner syndrome had at this time been attributed to monosomy of genes in the sex chromosomes, and narrowed down to the pseudoautosomal region. By studying patients with partial monosomy of this region and adults’ heights ranging from normal to more than 3 SD below average, the team refined this to a 170 kb section, in which they discovered SHOX.

Besides explaining the short stature of Turner syndrome patients, SHOX may also account for a small percentage of idiopathic short stature; the researchers discovered a mutation in one of 91 such patients tested.

And SHOX is just the tip of the iceberg, according to Dauber. He says that although the reviewed papers have provided “a tremendous amount of insight” into the GH/IGF-I axis, “there’s much more to growth than that one biological pathway.”

Illustrating this, Dauber points out that all reported patients with mutations in the GH/IGF axis number only a few hundred worldwide.

“But if you look at the number of kids who come to be evaluated by an endocrinologist, even with heights below minus 3 standard deviations or so, there are tens of thousands of those and we really only explain a tiny percentage of those with bona fide mutations in the GH/IGF axis.”

This suggests that most patients with idiopathic short stature will prove to have mutations in genes that are not directly involved in this pathway.

Not that Dauber believes the influence of the GH/IGF pathway is fully mapped out; he sees research in that area starting to focus more on mutations in regulatory regions of genes, and in epigenetics.

But he says: “I think that the challenge for endocrinologists in the coming years is going to be how to learn and think about the defects in growth that are outside that classic hormonal pathway.”

A complicating factor is the large grey area between polygenic short stature – caused by the cumulative effect of the thousands of variants with, at most, a millimetre effect on height – and monogenic short stature – caused by a mutation in a specific critical gene.

“The long-term challenge is going to be finding therapies that don’t affect the GH/IGF axis but do affect growth,” says Dauber.

Such therapies are coming into play, however; Dauber gives the example of the NPR2 gene, which encodes a receptor (natriuretic protein receptor B) that acts directly in the growth plate and is essential for endochondral ossification. An NPR2 agonist is currently in development for patients with achondroplasia, he says.

“That’s a totally different type of growth-promoting therapy, in a disorder that’s more commonly handled by geneticists than endocrinologists.”

Cincinnati Center for Growth Disorders, Division of Endocrinology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA

Martin Savage
Programme Director
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