GHR
Deficiency in Connection with Laron Syndrome
ABSTRACT
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.
INTRODUCTION
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.
Laron
Syndrome
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-/-.
RESULTS
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.
DISCUSSION
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.
Applications
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.
REFERENCES
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).
Aloha!!
