GHR Deficiency in Connection with Laron Syndrome
Growth hormone (GH) and insulin-like growth factor I (IGF-I) are essential components in the growth and development of an individual. Mutations in the GHR/BP gene cause a multitude of issues, including high levels of GH in serum and low levels of IGF-I in serum. These particular characteristics are unique to Laron syndrome, an autosomal hereditary recessive disorder. Other effects of this disease include retarded growth (dwarfism), lack of growth hormone binding protein (GHBP), and delayed puberty. A mammalian model for the disease other than humans was needed in order to advance in research, knowledge, and understanding of Laron syndrome because of ethical issues surrounding the use of humans as test subjects. Mice were utilized as potential candidates for research. The knockout mice were engineered to show the disrupted GHR/BP gene through use of a vector and homologous combination. The effects of the resulting mice that were homozygous and heterozygous for the mutation (GHR/BP-/- and GHR/BP +/-, respectively) were analyzed. The results of the knockout Laron mice (homozygous for the mutation) were compatible with characteristics shown by patients with Laron syndrome. Conclusions about insignificant differences between GHR/BP+/- mice and wild-type mice (GHR/BP+/+) were drawn, suggesting that only one functional GHR/BP allele is necessary for almost complete function of the gene. By studying Laron syndrome through knockout mice, potential applications including treatment of patients with the disease through biosynthetic IGF-I administration is a strong possibility. The success of the Laron knockout mouse promote further study and will help to solve many unresolved questions about the GHR/BP gene mutation and Laron syndrome in humans.
Background of Growth Hormone
Growth hormone (GH) is produced and secreted in the anterior of the pituitary gland, from where it then goes on to perform a multitude of biological functions including the promotion of growth. GH affects many types of tissues; its main role is to stimulate bone and soft tissue growth. Other responsibilities of GH include binding to growth hormone receptor (GHR) and internalization of the GH/GHR complex. This hormone/receptor complex is formed by a single molecule of GH that binds to two molecules of GHR. After the complex is activated, it signals stimulation of other genes, one of them being insulin-like growth factor I (IGF-I). IGF-I is a hormone and ligand that is mostly produced and secreted by the liver, but other target tissues may produce it as well. Its function is to mediate some of the indirect effects GH has on an organism’s growth and development. The function of IGF-I is continuous throughout an organism’s development. Growth hormone binding protein (GHBP) is a truncated or shortened form of GHR as it does not possess the transmembrane and intracellular regions. Instead, it corresponds to the extracellular domain of the GHR. The function of GHBP is not clear, but it seems to monitor the amount of growth hormone that circulates in the serum. GHBP is not produced the same way for all animal species. In mice and rats, alternative splicing of GHR precursor messenger RNA replaces the transmembrane and intracellular regions with a short hydrophilic tail (Coschigano, 2608). In humans, however, GHBP is made by proteolysis (the hydrolytic breakdown of proteins) of the GHR as opposed to alternative splicing. Both GHR and GHBP are encoded by a single GHR/BP gene, and are expressed in nearly all tissues of the body. The GHR/BP gene is encoded by 10 exons, with exon 4 coding for the GH binding domain. This is true for both humans and mice. Almost all of the functions of GH are accomplished due to its ability to interact with its receptor. The binding of GH to GHR leads to receptor dimerization (the chemical union of two identical molecules) and activation of a signal pathway that promotes growth.
In 1966, the first account of growth hormone resistance was described. Laron syndrome, also known as growth hormone insensitivity syndrome (GHIS), is caused by mutations of the GHR/BP gene for GHR. This variation of GHR leads to an insensitivity of growth hormone. There have been about 30 different types of inactivating mutations reported, including deletions, nonsense, missense, frameshift and splice, that affect the expression or function of both the GHR and GHBP. Mutations can reduce or inhibit dimerization of GHR once GH is bound. The mutation of the GHR/BP gene causes the GHR to become ineffective. Because of this, GH and GHR cannot communicate by signal transmission; therefore growth hormone can’t bind to its receptor. It has been found that most people diagnosed with Laron syndrome are from the Mediterranean or Middle Eastern regions, although some spontaneous mutations have been reported in other ethnic groups as well. Laron syndrome is a hereditary autosomal recessive disorder that is characterized by impaired growth even though levels of GH in blood serum remain normal or are even increased. This disorder is distinguished by short stature, or dwarfism, as well as facial dysmorphism, truncal obesity, delayed puberty, and recurrent hypoglycemia (Zhou, 13215). Those affected with Laron syndrome also show very high levels of GH in blood serum, very low levels of IGF-I in serum, and absent, low, or dysfunctional GHBP in serum. The high levels of GH are due to the fact that it cannot bind to GHR, so it simple continues to circulate in the serum. The reason IGF-I levels are low is because the active complex between GH and GHR are not formed, so there is no signal for IGF-I to be synthesized. Many features about the GHR mutation and Laron syndrome are unknown due to ethical issues surrounding the study of patients who have the disease. Therefore, an appropriate animal model of the disease would aid tremendously in determining all of the effects GHR and its mutation has on an individual.
MATERIALS AND METHODS
Use of Knockout Mice
To study Laron syndrome and the effects of the disorder, a mammal species would be the best candidate to use. However, there have been no reported mammals other than humans with a mutated growth hormone receptor. The only known animal that has been discovered exhibiting this mutation is the dwarf chicken, and is not an appropriate contender for the studies on Laron syndrome in humans as birds are very different from mammals in terms of anatomy and physiology. In addition to ethical issues, using human models for studying this disorder is impractical for identifying long-term effects because of the slow growth phase and long lifespan of people. A suitable model for Laron syndrome is the mouse with the correct knockout gene to mimic the disease in humans.
Creating the Mouse
One method of generating an animal that displayed the characteristics of Laron syndrome is to make the mouse resistant to growth hormone by expressing a GH antagonist gene. Another approach is to disrupt the mouse GHR/BP gene, which is essentially the defect that causes the disease in humans. In one study conducted by Zhou, Xu, and Maheshwari, the knockout mouse (Laron mouse) was created by disrupting the fourth exon and part of the fourth intron of the GHR/BP gene. This was done because exon 4 is where the binding domain for GH is, and also this is the location where mutations have been discovered in patients with Laron syndrome. Next, an EcoRI fragment was isolated from a mouse genomic library that included exon 4 of the mouse GHR/BP (mGHR/BP) gene. Then a targeting vector which held a neomycin resistance (neo) gene was created to replace/delete exon 4 and part of the fourth intron of the gene. Next, mouse embryonic stem (ES) cells were transfected with, or introduced to the newly created mGHR/BP targeting vector by electroporation. The knockout gene was integrated into the mouse genome by homologous recombination. Genomic DNA from the ES cells was then digested by BamHI, and the genotypes were identified by Southern blot analysis. The ES cells that were heterozygous for the disrupted GHR/BP gene (GHR/BP +/-) were then injected into blastocysts which were then transplanted into pseudopregnant mice. The embryos developed and the resulting mice were able to pass on the disrupted GHR/BP gene. Homozygous GHR/BP-disrupted (GHR/BP -/-) were the result of inbreeding of the F1 GHR/BP +/- mice. Southern blot analysis confirmed the results that mating of the F1 heterozygotes resulted in progeny that were GHR/BP+/+, GHR/BP+/-, and GHR/BP-/-.
Effect on Size
The physical effects of GHR/BP mutation are not directly evident, nor can they be measured and significantly interpreted between the GHR/BP+/+, GHR/BP+/-, or GHR/BP-/- after birth. After around three to four weeks of age, however, the weight of the Laron mouse (homozygous for the GHR/BP mutation: GHR/BP-/-) was considerable lower than the +/+ and +/- mouse. The +/- mice had an intermediate phenotype between those that were +/+ and -/- (Fig. 1). In terms of gender, the weights of males and females in both +/+ and +/- mice were notably different, but the weights of -/- mice were statistically irrelevant when comparing males to females. This indicates a loss of gender difference in the -/- mice (Coschigano, 2609). Overall, -/- mice grew much slower and reached their maximum weight earlier than the +/+ and +/- mice. The differences in weight between -/- and +/+, +/- mice increased progressively with age.
Effect on GH, IGF-I, GHBP
Other characteristics of Laron mice were observed as well. The level of GH in blood serum in GHR/BP-/- mice were significantly higher than those of the GHR/BP+/+ and GHR/BP+/- genotype. The levels of GH between +/+ and +/- mice were similar. For these results, there were no major differences between the male and female mice of any type. The high levels of GH in the blood was due to the mutated GHR and lack of signal transmission, which inhibited the binding of the GH/GHR complex and left growth hormone free to circulate in the serum. In contrast to increased levels of GH, there were diminished levels of IGF-I by approximately 90% in -/- serum. IGF-I levels in +/+ and +/- mice were, again, not statistically significant. Again, no important distinctions were found between males and females. The decrease of IGF-I levels was a result of the active complex between GH and GHR not forming. Therefore, no signal to synthesize IGF-I was produced (Fig. 2).
Fig. 1 (above). Phenotypic size differences between
GHR/BP+/+, GHR/BP+/-, and GHR/BP-/- female mice at 5 months. Left, wild type (+/+). Middle, homozygous for the GHR/BP gene mutation (-/-). Right, heterozygous for the GHR/BP gene mutation (+,-) (Coschigano, 2609).
Fig. 2 (right). Concentrations of GH (A) and IGF-I (B) levels in blood serum for the +/+, +/-, and -/- mice. Average results from 3-4 mice of each genotype at ages 30 and 60 days (Zhou, 13219).
Along with low IGF-I levels reported, IGF binding protein (IGFBP) was also evaluated for the effect that the GHR/BP gene mutation has on it. No differences were seen among IGFBP-1, IGFBP-2, or IGFBP-4 levels in the -/- mice. However, IGFBP-3, the principal carrier protein for IGF-I, was greatly reduced when compared to the level of IGFBP-3 in +/+ mice. Levels of all IGFBPs in +/- mice were comparable to those of the +/+ genotype. GHBP was not identified in the serum of -/- mice. The wild type (+/+) displayed normal levels of GHBP, but those that were heterozygous for the GHR/BP disruption had slightly decreased levels.
Effect on Sexual Maturation
The average litter size of GHR/BP+/- mice (similar to +/+) vs. that of GHR/BP-/- mice was 6.57: 2.71, respectively. Furthermore, the mortality rate of newborns from inbred -/- progeny was significantly higher than the +/+ or +/- genotypes. This may be a result of several different factors, including maternal-fetal size mismatch and inadequate lactation of the mothers to feed their pups adequately. In addition to litter size and mortality rate of pups, a delay in first pregnancy of -/- mice was observed, implying that sexual maturation is delayed in females (Zhou, 13217).
Effect on Longevity
The lifespans of each genotype (+/+, +/-, -/-) and gender were analyzed for the purpose of gauging longevity. The hypothesis was that decreased body size increased lifespan of an individual. The results showed an increase in lifespan of nearly 40% in those homozygous for the GHR/BP gene mutation. The -/- mice live, on average, almost an entire year longer than the +/+ and +/- counterparts, who showed no significant difference in lifespan themselves. More research needs to be done in this area with knockout mice to determine what exactly causes the increased lifespan of those with the disrupted GHR/BP gene, or those with smaller body size in general.
Functionality of GHR/BP Allele
The results of the knockout mice heterozygous for the GHR/BP gene mutation were noteworthy. Most of the results, including serum IGF-I levels, serum GH levels, sexual maturation, and longevity showed insignificant differences between the +/+ and +/- genotypes. These outcomes suggest that the loss of one allele for the GHR/BP gene (heterozygous individuals) has little to no effect on the gene functioning in a normal manner. Loss of both alleles (-/- mice), however, results in drastic changes to the phenotype. Therefore, the observation of the slight differences between +/+ and +/- mice implies that only one functional allele for the GHR/BP gene is needed to almost completely express its activity to the fullest potential.
Knowledge concerning Laron syndrome is an important field of study in determining how to treat patients with the disorder. An example of such a treatment is administering biosynthetic IGF-I to children. This method of treatment stimulates growth and appeared to regulate biochemical abnormalities. One of the unanswered questions with this technique, however, is whether this treatment is safe and if it could reverse changes that were caused by long-term deficiency of IGF-I. A possible safety issue is potential overdose on IGF-I treatment which can lead to complications such as hypoglycemia and edema (Laron, 4397). These effects are supposedly reversible by lowering the dosage of IGF-I. A useful means of controlling the amount of IGF-I treatment given to a patient is monitoring IGF-I levels in serum. With further study, this treatment may become essential in treating patients with Laron syndrome.
Success with the Laron Mouse
The results from the knockout Laron mice discussed above are analogous to results found in humans with Laron syndrome. The most prominent features that occur in both knockout Laron mice and people with the disease in physical and biochemical terms include high levels of GH, low levels of IGF-I, nonexistent amount of GHBP, growth retardation, and delayed sexual maturation. This signifies that mice are a good model to use to study the GHR/BP mutation and its effects. It was imperative that a suitable substitute was found in order to make advancements on knowledge of this disorder and its consequences. Humans were not viable candidates as there are limitations on the types of tests that can be performed due to ethical reasons. It was also important that a mammal be used to mimic the effects of Laron syndrome in order to compare the results obtained to humans who possess the disease. When considering the effect this gene disruption has on longevity and other long-term consequences, it is crucial to use an animal model with a rapid growth rate and short lifespan in order to avoid limitations based on time restraint. Along with researching longevity, using a mouse model will allow for research on body composition and tissue characteristics on individuals that possess the GHR/BP gene mutation that wasn’t able to be performed before. The success of the knockout Laron mouse will prove to be helpful in discovering answers to many unresolved questions about Laron syndrome.
Coschigano, Karen T., David Clemmons, Linda L. Bellush, and John J. Kopchick. "Assessment of Growth Parameters and Life Span of GHR/BP Gene-Disrupted Mice." Endocrinology 141.7 (2000): 2608- 613. Print.
Laron, Zvi. "The Essential Role of IGF-I: Lessons from the Long-Term Study and Treatment of Children and Adults with Laron Syndrome." Journal of Clinical Endocrinology & Metabolism 84.12 (1999): 4397-404. Print.
Zhou, Yihua, Bixiong C. Xu, and Hiralal G. Maheshwari, et al. "A Mammalian Model for Laron Syndrome Produced by Targeted Disruption of the Mouse Growth Hormone Receptor/binding Protein Gene (the Laron Mouse)." Proceedings of the National Academy of Sciences of the United States of America 94.24 (1997): 13215-3220. Print.
....Holy shit, right? Most scientific thing I've ever written in my life. And I'm damn proud of it too!
Now who wants to proofread this and edit it for me? You know your life sucks when you write a paper you don't even want to read yourself.
Gotta love end of the semester projects, papers, and... finals!
"Deck the dorms with cups of coffee - fa-lalalala-lala-la-la
Tis the season to....eat toffee - fa-lalalala-lala-la-la
Don we now our baggy eyelids - fa-lala-lalala-la-la-la!!!!
Trolls, we are, us dumb college kids - fa-lalalalaaaa-lalaaaa-laaaa-laaaaaaaaaaa"
Okay but seriously it's two in the morning and I've never stayed up this late on a school night in college. Ever. But now I can't say that.
Between the paper writing and being awake at this ungodly hour, I think it's time for me to hit the sack.
I'll make another more productive post sometime soon. In the meantime, if you need a leisurely read, knock your socks off and have a go with my genetics paper (if you haven't indulged already).