Chapter 1a – Growth and Growth Disorders

Berrin Ergun-Longmire, MD, Assistant Professor of Pediatrics, NEOUCOM, Akron Children's Hospital, Center for Diabetes and Endocrinology, 215 West Bowery Street, Suite 6400,
Akron, Ohio 44308-1062

Michael P. Wajnrajch, MD Clinical Associate Professor, New York University, Department of Pediatrics, 550 1st Avenue, New York, NY 10016
Senior Medical Director, Pfizer Global Pharmaceuticals, 235 East 42nd Street, MS 219-07-01
New York, NY  10017

Updated January 4, 2010

INTRODUCTION

Human growth starts at conception and proceeds through various identifiable developmental stages. The process of growth depends on both genetic and environmental factors that combine to determine an individual's eventual height. The genetic control of statural growth is becoming increasingly clear. Many genes have been identified that are required for normal development and function of the pituitary in general, and that control the growth hormone/insulin-like growth factor axis in particular. Mutations of these genes have been shown to be responsible for abnormal growth in humans and animals.

Growth hormone has been used to treat short children since the 1950s. Initially only those children with the most pronounced growth failure due to severe growth hormone deficiency (GHD) were considered appropriate candidates, but with time children with growth failure from a range of conditions have been shown to benefit from GH treatment. GH has also been used to treat several catabolic processes, including cystic fibrosis, inflammatory bowel disease and AIDS wasting. Here we review the physiology of growth, the diagnosis of GH deficiency, treatment options and genetic growth hormone disorders.

GROWTH DISORDERS

Growth failure may be due to genetic mutations, acquired disease and/or environmental deficiencies. Growth failure may result from a failure of hypothalamic GHRH production or release, from (genetic or sporadic) maldevelopment of the pituitary somatotropes, secondary to ongoing chronic illness, malnutrition, intrinsic abnormalities of cartilage and/or bone such as osteochondrodysplasias, and from genetic disorders affecting growth hormone production and responsiveness. Children without any identifiable cause of their growth failure are commonly labeled as having idiopathic short stature.

Genetic factors affecting growth include pituitary transcription factors (PROP1, POU1F1, HESX1, LHX3, and LHX4) as well as hormones (growth hormone-releasing hormone (GHRH), GH secretagogues (GHS), GH, insulin like growth factor-1 (IGF1), insulin like growth factor-2 (IGF2), insulin (INS)) and their receptors (GHRHR, GHSR, GHR, IGF1R, IGF2R and INSR) as well as transcription factors controlling GH signaling, including STAT1, STAT3, STAT5a, and STAT5b. Growth is also influenced by other factors such as sex steroids (estrogens and androgens), glucocorticoids and thyroid hormone.

Since the replacement of human pituitary-derived GH with recombinant human GH, much experience has been gained with the use of GH therapy. The Food and Drug Administration (FDA) had expanded GH use for the following conditions for children (1):

1.     GH deficiency/insufficiency

2.     Chronic renal insufficiency (pretransplantation)

3.     Turner syndrome

4.     SHOX haploinsufficiency

5.     Short stature from Prader-Willi Syndrome (PWS)

6.     Children with a history of fetal growth restriction (SGA, IUGR) who have not caught up to a normal height range by age 2 years

7.     Children with idiopathic short stature (ISS): height > 2.25 SD below the mean in height and unlikely to catch up in height.

8.     Noonan Syndrome

FDA approved conditions for GH treatment for adults:

1.     Adults with GH deficiency

2.     Adults with AIDS wasting

The efficacy of GH treatment has been investigated in children whose height has been compromised due to chronic illnesses such as Crohns disease, cystic fibrosis, glucocorticoid-induced suppression of growth in other disorders (asthma and juvenile idiopathic arthritis (JIA), also known as juvenile rheumatoid arthritis (JRA)), and adrenal steroid disorders such as congenital adrenal hyperplasia (CAH). Studies have shown both anabolic effects and improvement of growth velocity after GH treatment in children with glucocorticoid dependent Crohns disease (2-4). Improvement in linear growth has also been observed after GH treatment in children with cystic fibrosis and JIA (5-7). The same studies have shown significant improvement in weight gain and body composition, changes that have been variably correlated with improvement in life expectancy and quality of life.

The growth-suppressing effects of glucocorticoids, is also seen in children affected with CAH where high androgens limit the height potential. Most patients with CAH complete their growth prematurely and are ultimately short adults. Lin-Su et al., showed that GH in combination with LHRHa significantly improved their final adult height in children with CAH (8).

Larger, long-term prospective studies are needed to determine the safety and efficacy of GH treatment in these populations of children.

The key mediator of GH action in the periphery for both prenatal and postnatal mammalian growth is IGF system. GH exerts its direct effects at the growth plate and indirect effects via IGF1. Better understanding the role of IGF1 on growth had led to the concept of IGF1 deficiency in addition to GH deficiency. With the introduction of recombinant human (rh)IGF1, today, it is possible to treat conditions due to genetic GH resistance or insensitivity caused by GH receptor defects, and the presence of neutralizing GH antibodies (154).

DIAGNOSIS

Diagnosis of GH deficiency during childhood and adolescence is frequently challenging. Children whose height are below the 3rd percentile or -2 SD and have decreased growth velocity require clinical evaluation. Evaluation should begin with a detailed past medical history, family history, diet history, detailed review of prior growth data (including the initial post-natal period) and a thorough physical examination (9). Together, these should help the clinician identify the pattern and cause of growth failure, such as fetal growth restriction (e.g. SGA and IUGR), chronic illness, malnutrition/malabsorption, hypothyroidism, skeletal abnormalities or other identifiable syndromes, such as Turner syndrome. Once growth hormone deficiency is suspected, further testing of the hypothalamic-pituitary axes (including but not limited to the GH-IGF axis) along with radiological evaluation, should be performed (Table 1). It is important to note that the tests cannot be performed simultaneously, or in random order. Certain conditions (e.g. Hypothyroidism and Celiac disease) may mask the presence of others (e.g. GH deficiency), therefore requiring to a step-wise approach with screening tests preceding specific examinations. Since growth failure generally occurs outside of GHD, only those children with signs or symptoms undergo expensive, invasive and nonphysiologic GH provocative testing.

Table 1. Guidelines for initial clinical evaluation of a child with growth failure
Evaluation	Key elements
Birth history	Gestational age, birth weight and length, delivery type, birth trauma, hypoglycemia, prolonged jaundice.
Past medical and surgical history	Head trauma, surgery, cranial radiation, CNS infection.
Review of systems	Appetite, eating habits, bowel movements.
Chronic illness	Anemia, Inflammatory Bowel Disease, cardiovascular disease, renal insufficiency, etc.
Family history	Consanguinity, parents and siblings' heights, family history of short stature, delayed puberty.
Physical examination	Body proportions (upper/lower segment ratios, arm span), head circumference, microphallus, dysmorphism, and midline craniofacial abnormalities.
Growth pattern	Crossing of percentiles, failure to catch-up.
Screening Tests	CBC, BCP, ESR, TFT's, UA, IGF1, IGFBP3, Bone age (and a Karyotype for females)

Growth charts:

The growth pattern is a key element of growth assessment and is best studied by plotting growth data on an appropriate growth chart. US growth charts were developed from cross-sectional data provided by the National Center for Health Statistics and updated in 2000 (10), with body mass index included in this newest set. The supine length should be plotted for children from birth through age 3 years and standing height plotted when the child is old enough to stand, generally after 2 years of age. Ideally, growth data is determined by evaluating subjects at regular (optimally at 3 month) intervals, with the same stadiometer, and with the same individual obtaining the measurements, whenever possible. Three months is the minimal time interval needed between measurements to calculate a reliable growth velocity, and a six to twelve month interval is optimal. Age and pubertal staging must be considered when evaluating the growth velocity, with the understanding that there is great individual variation in the onset and rate of puberty (11).

Deviations across height percentiles should be noted and evaluated further when confirmed, with the understanding that during the first two years of life, the crossing of length and/or weight percentiles may reflect catch-up or catch-down growth. Crossing percentiles during this period is not always physiological, and must be examined in the context of family, prenatal, birth and medical histories. Additionally, between two and three years of age, statural growth measurement changes from supine to erect, and may also introduce variation. Growth below the normal range (e.g. >-2SD) even without further deviation is consistent with (but not pathognomonic of) GH deficiency. Short stature with a low BMI suggests an abnormality of nutrition/GI tract (e.g. malnutrition, Celiac Disease, etc.), while short stature with an elevated BMI suggests hypothyroidism, Cushing's syndrome or a central eating disorder, such as Prader-Willi syndrome, etc.

Figures 1-3 represent growth charts of children studied by the authors who have genetic defects leading to isolated growth hormone deficiency.

Figure 1. Growth pattern in children with isolated GH deficiency (Type 1A)

Growth failure can manifest as either gradual deceleration ("falling off the curve") (Figure 2), or alternatively, maintaining a growth pattern parallel to the 3rd percentile, but without catch-up growth (Figure 3).

Figure 2. Growth pattern in children with isolated GH deficiency (Type 1B)

Figure 3. Growth pattern in children with isolated GH deficiency (Type 2)

Most children with GH deficiency have normal birth weight and length. However, in most cases, postnatal growth becomes severely compromised. This can be seen even in the first months of life. Although such children may show a normal growth pattern during the first 6 months, growth failure will eventually occur, as GH takes on a more physiologically dominant role and a child's growth falls below the normal range.

Radiologic evaluation:

The most commonly used system to assess skeletal maturity is to determine the 'bone age' of the left hand and wrist, using the method of Greulich and Pyle (12). Children younger than 2 years of age should have their bone age estimated from x-rays of the knee. Tanner and Whitehouse and their colleagues developed a scoring system for each of the hand bones as an alternative method to the method of Greulich and Pyle (13).

Adult height prediction methods estimate adult height by evaluating height at presentation relative to normative values for chronological or bone age. Such methods have been utilized for approximately 60 years (14) and are generally considered accurate in evaluating healthy children with a normal growth potential (15, 16). Several different methods have been produced and are currently in widespread use, including those of Bayley-Pinneau, the Tanner-Whitehouse-Marshall-Carter and Roche-Wainer-Thissen.

In 1946, Bayley initially described how final height could be estimated from the present height and the bone age, revising the method in 1952 to use the bone age assessment method of Greulich and Pyle (17). They developed what is commonly known as the predicted adult height (PAH) method of Bayley-Pinneau (BP). Tables have been developed for the BP method, listing the proportion of adult height attained at different bone ages, using longitudinal growth data on 192 healthy children in the US. Three tables average, advanced and retarded correct for possible differences between CA and BA of more than one year (18). The Bayley-Pinneau PAH method is applicable from age 8 years onwards.

Tanner, Whitehouse, Marshall and Carter developed an adult height prediction model based on current height, the mid-parental height, the age of menarche in girls and the Tanner bone age (13). This PAH method (TW2) was developed on the longitudinal data of 211 healthy, British children. TW2 differs from the BP method in that the TW2 lowers the minimal age of prediction to 4 years; and also allows for a quantitative effect of BA, while BP give a semi-quantitative effect of bone age (i.e. delayed, normal or advanced).

The PAH method of Roche-Wainer-Thissen (RWT) was derived from longitudinal data on approximately 200 normal Caucasian American children in southwestern Ohio, at the Fels Research Institute (19). The RWT PAH method assesses the subjects height, weight, BA and mid-parental height (MPH) and then applies regression techniques to determine the mathematical weighting to be applied to the four variables. The RWT method was designed to allow final height prediction from a single visit, but is only applicable when greater than half of the bones are not fully mature.

Since both the bone age assessments and height prediction methods are created from healthy children (and often children from a single ethnic group and region), their use in other populations is potentially inappropriate. In fact, Tanner et al state that their method is applicable to both boys and girls with short stature, but caution that In clearly pathological children, such as those with endocrinopathies, they do not apply. Similarly, Roche et al suggest caution when applying the RWT PAH method in non-white and pathological populations (19). Zachmann et al reported that the RWT and TW2 methods (which are more BA-reliant) are better when growth potential is normal relative to the BA, however, in conditions with abnormal and incorrigible growth patterns the BP method was more accurate, stating that with a non-normal bone maturation to growth potential relationship, the coefficient and regression equations (RWT and Tanner) cause an over-prediction of adult height (20).

As stated above, these methods are based on healthy children and assume that the growth potential is directly proportional to the amount of time left prior to epiphyseal fusion as measured by the bone age. While this is correct for some of the children seen by the pediatric endocrinologist (e.g. healthy children, children with GH deficiency), it is not correct for many others with abnormal growth (e.g. children born SGA, children with idiopathic short stature, Turner syndrome and chronic renal failure). It is likely also inappropriate for children with an abnormal tempo of maturation (e.g. children with Russell-Silver syndrome, precocious puberty and congenital adrenal hypertrophy). In such children, standard growth prediction methods should be used only as general guides, if at all.

Biochemical evaluation of GH deficiency:

As growth hormone is secreted in a pulsatile manner (usually 6 pulses in 24 hours and mainly during the night) with little serum GH at any given time, several methods have been recommended to assess the adequacy of GH secretion:

1.     Stimulation testing: GH provocation utilizing arginine, clonidine, glucagon, L-Dopa, insulin, etc. This practice generally measures pituitary reserve-or GH secretory ability-rather than endogenous secretory status. Trained individuals should perform the GH stimulation test according to a standardized protocol, with special care taken with younger children/infants.

2.     GH-dependent biochemical markers: IGF1 and IGFBP3: Values below a cut-off less than -2 SD for IGF1 and/or IGFBP3 strongly suggest an abnormality in the GH axis if other causes of low IGF have been excluded. Age and gender appropriate reference ranges for IGF1 and IGFBP3 are mandatory.

3.     24-hour or Overnight GH sampling: Blood sampling at frequent intervals designed to quantify physiologic bursts of GH secretion.

4.     IGF generation test: This test is used to assess GH action and for the confirmation of suspected GH insensitivity. GH is given for several days (3-5 days) with serum IGF1 and IGFBP-3 levels measured at the start and end of the test. A sufficient rise in IGF1 and IGFBP-3 levels would exclude severe forms of GH insensitivity (11, 21).

Failure to raise the serum GH level to the threshold level in response to provocation suggests the diagnosis of GH deficiency, while a low IGF1 and/or IGFBP3 level is supportive evidence. GH stimulation test results should be interpreted carefully in conjunction with pubertal status and body weight. Puberty and administration of the sex steroids increase GH response to stimulation tests (155). To prevent false positive results, some centers use sex steroid priming in prepubertal children prior to GH stimulation testing (156). In obese children, the normal regulation of the GH/IGF1 axis is disturbed and GH secretion is decreased. In addition, IGF1 levels are very sensitive to the nutritional status (IGFBP3 less so), and also that the normative range for IGF1 and IGFBP3 values are extremely wide, often with poor discrimination between normal and pathological. Age/pubertal stage and gender-specific threshold values must be utilized for both IGF1 and IGFBP3, i.e. GH threshold of 10 ng/ml in children, 5 ng/ml in older adults, etc.

Summary of diagnosis of GH deficiency:

Children with severe GH deficiency can usually be diagnosed easily on clinical grounds, and fail GH stimulation tests. Studies have shown that despite clinical evidence of GH deficiency, some children may pass GH stimulation tests (11). In the case of unexplained short stature, if the child meets most of the following criteria, a trial of GH treatment should be initiated (1):

1.     height >2.25 SD below the mean for age or >2 SD below the midparental height percentile;

2.     growth velocity <25th percentile for bone age:

3.     bone age >2 SD below the mean for age;

4.     low serum insulin-like growth factor 1 (IGF1) and/or insulin-like growth factor binding protein 3 (IGFBP3);

5.     other clinical features suggestive of GH deficiency.

Key elements that may indicate GH deficiency are:

1.     Height more than 2 SD below the mean.

2.     Neonatal hypoglycemia, microphallus, prolonged jaundice, or traumatic delivery.

3.     Although not required, a peak GH concentration after provocative GH testing of less than 10 ng/ml.

4.     Consanguinity and/or a family member with GH deficiency.

5.     Midline CNS defects, pituitary hypo- or aplasia, pituitary stalk agenesis, empty sella, ectopic posterior pituitary (bright spot) on MRI.

6.     Deficiency of other pituitary hormones: TSH, PRL, LH/FSH and/or ACTH deficiency.

Many practitioners consider GH stimulation tests to be optional in the case of clinical evidence of GH deficiency, in patients with a history of surgery or irradiation of the hypothalamus/pituitary region and growth failure accompanied by additional pituitary hormone deficiencies. Similarly children born SGA, with Turner syndrome, PWS and chronic renal insufficiency do not require GH stimulation testing before initiating GH treatment (1).

TREATMENT

The principal objective of GH treatment in children with GH deficiency is to improve final adult height. Human pituitary-derived GH was first used in children with hypopituitarism over 50 years ago, and abruptly ceased in 1985, after the first cases of Creutzfeld-Jacob disease were recognized. Since 1986, recombinant human GH (rhGH) has been the exclusive form of growth hormone used to treat GH deficiency in the United States and most of the world.

Short stature without overt growth hormone deficiency is very well described, and occurs in Turner Syndrome, renal failure, malnutrition, cardiovascular disease, Prader-Willi syndrome, small for gestational age, inflammatory bowel disease and osteodystrophies-and clearly represents the majority of short/poorly growing children in the world. Although not the focus of this discussion, it is important to realize that-in clinical terms-GH therapy is used to treat growth failure, rather than a biochemical GH deficiency. GH therapy in this setting, in combination with disease-specific treatments, generally improves statural growth and final adult height.

The primary goals of the treatment of a child with GH deficiency are to achieve normal height during childhood and to attain normal adult height for children with GH deficiency. Children should be treated with an adequate dose of rhGH, with the dose tailored to that childs specific condition. FDA guidelines for GH dose vary according to the indication and are given in Table 2 (1).

Administration of rhGH in the evening is designed to mimic physiologic hGH secretion. Treatment is continued until final height or epiphyseal closure (or both) has been recorded. GH therapy, however, should be continued throughout adulthood in the case of GHD, to optimize the metabolic effects of GH and to achieve normal peak bone mass-albeit at significantly lower "adult" doses. Adult GH replacement should only be started after retesting the individual and again demonstrating a failure to reach the new age-appropriate GH threshold, if appropriate.

Table 2. GH dosage.
Indication	Dose (mg/kg/wk)
GH Deficiency   Children Pre-pubertal Pubertal Adults	0.16 - 0.35 0.16 - 0.70 0.04 - 0.175
Turner Syndrome	0.375
Chronic renal insufficiency	0.35
Prader-Willi Syndrome	0.24
SGA	0.48
Idiopathic short stature (ISS)	0.3 0.37

The growth response to GH treatment is typically maximal in the first year of treatment and then gradually decreases over the subsequent years of treatment. First year growth response to rhGH is generally 200% of the pre-treatment velocity, and after several years, averages 150% of the baseline. Height improvements of 1 SD are typically achieved in children with GHD after two years of treatment, and between 2 and 2.5 SD after five or seven years.

GH doses are often increased if catch-up growth is inadequate and/or to compensate for the waning effect of rhGH with time. A recent report showed a significant improvement in HV when GH dose was adjusted based on IGF1 levels (157). However, GH dose was almost 3 times higher than mean conventional GH dose when IGF1 levels were titrated to the upper limit of normal. The lack of long-term safety data on high doses of GH and high circulating levels of IGF1 levels should be considered. Therefore, weight-based GH dosing is still recommended by many as the standard of care (158).

It is critically important to maximize height with GH therapy before the onset of puberty. Several investigators have advocated modifying puberty or the production of estrogens by the use of GnRH super-analogues (22, 23) and aromatase inhibitors (24-27), respectively, in order to expand the therapeutic window for GH treatment, especially in older males.

The response to GH may vary in children if they are not GH deficient (i.e., SGA, Turner syndrome). Recent reports indicating an association of the deletion of Exon 3 on GH receptor.

Monitoring GH Treatment

Children receiving GH therapy require periodic monitoring. Three-month intervals are commonly chosen to allow for sufficient growth for a meaningful measurement, while minimizing time between dose adjustments/intervention. During follow up visits, height, weight, pubertal status, inspection of injection sites, and a comprehensive clinical exam should be initiated. In clinical practice, there are several parameters to monitor the response to GH treatment; the determination of the growth response (i.e. change in height velocity, ∆HV) being the most important parameter.

Table 3. Summary of follow-up evaluation.
Parameters	Assessment
Bone age	12 month intervals to assess the predicted height.
Thyroid Function Test	6 month intervals, or immediately, if growth velocity decreases.
Serum IGF1 and IGBP-3	3-12 month intervals. Most useful in maintaining GH dose in 'safe' region. They do not necessarily correlate with growth velocity.
Metabolic panel, CBC,ESR, HbA1C	12-month intervals.
Dose adjustment	Weight-adjustment, growth response, pubertal stage, IGF level and comparison to predicted height.
Adverse Events	Every visit.

The Safety of GH Treatment

To date, multiple studies have demonstrated the safety of GH therapy (28-31)(7-10). Patients, however, should be monitored closely during treatment. While rhGH treatment is generally considered safe, concerns have been raised about tumorigenicity of chronically elevated IGF1 levels. It would therefore seem prudent to maintain IGF1 levels in the mid-normal range for age/pubertal stage and gender. Although the long-term consequences of elevated IGF1 levels during childhood are not known, some investigators recommend that dose reductions be considered after the first two years of therapy if IGF1 levels continue to be above the normal range (1).

Another concern is the use of GH in patients with Prader-Willi syndrome. Early recognition of the syndrome allows earlier intervention to prevent morbidity. Previous studies and data from KIGS showed that earlier initiation of GH treatment in children with PWS significantly improved body composition, growth, and cognition (159).

To date, a total of 28 fatalities have been reported in patients with Prader-Willi syndrome during or after rhGH therapy (160). Data for children aged 3 years and older showed no statistically significant differences between the GH-treated and untreated groups with respect to cause of death, including respiratory infection or insufficiency (32, 160). Although there is no clear evidence that those deaths are related to GH therapy, it was postulated that GH/IGF1 may worsen sleep apnea or hypoventilation via increasing tonsillar/adenoid tissue or worsen pre-existing impaired respiration by increasing volume load (33). However, studies on respiratory function of subjects with Prader-Willi syndrome during rhGH therapy have only demonstrated improved respiratory drive and function (34). In fact, a recent study showed that all subjects tested had abnormal sleep studies/parameters prior to initiating GH treatment, and that GH treatment resulted in an improvement in sleep apnea in the majority of patients with PWS. Importantly, however, a subset had worsening of sleep disturbance shortly after (6 wk) starting GH when also developing a respiratory infection (35). Because it is difficult to predict who will worsen with GH treatment, these authors recommend that patients with Prader-Willi syndrome have polysomnography before and 6 wk after starting rhGH and should be monitored for sleep apnea during upper respiratory tract infections. IGF1 levels should also be monitored.

The data on efficacy and safety of GH treatment in 5220 Turner Syndrome (TS) children during the last 20 years has been reported by NCGS. The incidence of various side effects known to be associated with GH including pseudotumor cerebri, slipped capital femoral epiphysis, and scoliosis was increased in TS patients treated with GH compared with non-TS patients, however, children with TS are known to have a higher incidence of these side effects independent of rhGH treatment (161). Interestingly, type 1 diabetes was increased in GH treated group, most likely unrelated to GH treatment since the predisposition to autoimmune disorders is one of the characteristics of TS. In addition, NCGS data demonstrate a slightly increased incidence of a variety of malignancies in TS, however, this may again be related to the underlying condition, (i.e. not necessarily the rhGH treatment) as girls with TS have been shown to have an increased risk for cancer compared to general population (162.). In summary, twenty years of experience in 5220 patients seems reassuring and does not indicate any new rhGH-related safety signals in the TS population (161).

The common side effects of GH therapy are summarized in Table 4.

Table 4. Real and Theoretical Side Effects of GH treatment
Side effects	Comment
Slipped capital femoral epiphysis (SCFE)	Unclear whether GH causes SCFE or if it is a result of diathesis and rapid growth, induced by the GH. Should be evaluated at each visit for knee or hip pain/limp
Pseudotumor cerebri	The mechanism is unclear, but it may be a result of GH induced salt and water retention within the CNS. Complaints of headache, nausea, dizziness, ataxia, or visual changes should be evaluated immediately
Leukemia	Numerous large studies have not shown any association between rhGH and leukemia in children without predisposing conditions (28, 36)
Insulin resistance	Patients with a limited insulin reserve may develop glucose intolerance. HbA1c should be monitored
Recurrence risk of CNS tumors	Extensive studies did not support this possible side effect (37, 38)
Hypothyroidism	Almost 25 % of children may develop declines in serum T4 levels, generally reflecting enhanced conversion of T4 to T3, rather than outright hypothyroidism
Transient gynecomastia	These are attributed to anabolic and metabolic effects of GH
Scoliosis	Follow for progression and refer as appropriate
Sleep apnea/sleep disturbance	GH treatment might worsen sleep apnea/sleep disturbance in patients with Prader-Willi Syndrome, especially during a concomitant respiratory infection.

MULTIPLE PITUITARY HORMONE DEFICIENCY (MPHD)

GH deficiency may occur in combination with other pituitary hormone deficiencies and is often referred to as hypopituitarism, panhypopituitarism or multiple pituitary hormone deficiency (MPHD).

The anterior portion of pituitary gland forms from Rathke's pouch around the third week of gestation (45). A number of transcription factors have been identified that are required for normal pituitary development, while others are required for continued normal function (Table 4). Mutations have been identified in the genes for several of these pituitary transcription factors, including LHX3, LHX4, RPX1, PROP1 and POU1F1.

Table 5. Transcription factors required for normal pituitary development.
Transcription Factors	Function
1. LHX3	Essential for the early development of the anterior pituitary, including the somatotrope, thyrotrope, lactotrope and the gonadotrope (but not the corticotrope)
2. LHX4	Essential for the proliferation of the anterior pituitary cell types, including the somatotrope, thyrotrope and the corticotrope
3. HESX1	Essential for the development of the anterior pituitary, including the somatotrope, thyrotrope, lactotrope and the gonadotrope
4. PROP1	Essential for the development of most cell types of the anterior pituitary, including the somatotrope, the thyrotrope, the lactotrope and the gonadotrope (but not the corticotrope). Also essential for the expression of the PIT1 protein and the extinction of HESX1 in the anterior pituitary
5. POU1F1 (Pit1)	Necessary for somatotrope, lactotrope and thyrotrope development and also for their continued function
6. SOX3	Essential for the early formation of hypothalamic-pituitary axis

LHX3 is a member of the LIM family of HomeoboX transcription factors. The gene LHX3, is located on 9q34.3, comprises 7 exons (including two alternative exon 1's, 1a and 1b) and encodes a protein of 402 amino acids (Figure 4). LHX3 is expressed in the developing Rathke's pouch and is required for the development of most anterior pituitary cell types, including the somatotrope, the thyrotrope, the lactotrope and the gonadotrope (but notably not the corticotrope). LHX3 binds as a dimer, synergizing with POU1F1 (PIT1). Two unrelated families with MPHD were identified in 2000 (46) as harboring mutations in LHX3. The affected members of the family manifested severe growth retardation in association with restricted rotation of the cervical spine. Inheritance is consistent with an autosomal recessive pattern of inheritance and of note one individual was found to have an enlarged pituitary. Recently, a new mutation in LHX3 was described in a child with hypointense pituitary lesion, focal amyotrophy and mental retardation in addition to neck rigidity and growth retardation (47). These clinical findings expand the phenotype associated with mutations in LHX3.

Figure 4.

LHX4 also has a critical role in the development of anterior pituitary cells, and is co-expressed with LHX3 in Rathke's pouch in an overlapping but not wholly redundant pattern. Raetzman et al showed overlapping functions with Prop1 in early pituitary development, but also observed that their mechanisms of action were not identical. Lhx4 is necessary for cell survival and Lhx3 expression, with the pituitary hypoplasia seen in Lhx4 mutants actually results from increased cell death and reduced differentiation, directly attributable to loss of Lhx3; while Prop1 mutants exhibit normal cell proliferation and cell survival but show evidence of defective dorsal-ventral patterning (48). In the absence of both of these genes, no specification of corticotropes, gonadotropes or thyrotropes occurs in the anterior lobe. Although both LHX3 and LHX4 are crucial for the development of pituitary gland, LHX3 is expressed at all stages studied, whereas LHX4 expression is transient at 6 weeks of development (49). LHX4 is located on 1q25 and comprises 6 exons spread over a 45 kb genomic region (Figure 5). An intronic splice site mutation has been described in one family, manifesting GH, TSH and ACTH deficiency, along with cerebellar and skull defects. The mutation is transmitted as an autosomal dominant condition, with complete penetrance. Interestingly, a heterozygous mutant mouse model had no discernable phenotype, while homozygous loss of function in the mouse was fatal (50).

Figure 5.

RPX1 (Rathke's Pouch HomeoboX) is also referred to as HESX1 (HomeoboX gene expressed in Embryonic Stem cells) and is necessary for the development of the anterior pituitary. RPX1 comprises 4 exons and encodes a protein of 185 amino acids that features both a homeodomain as well as a repressor domain and is located on chromosome 3p21.2 (Figure 6). The extinguishing of HESX1 requires the appearance of another pituitary transcription factor, PROP1. A mutation has been described in two children of a consanguineous union who had optic nerve hypoplasia, agenesis of the corpus callosum and panhypopituitarism, with an apparent autosomal recessive mode of inheritance (51). This Arg --> Cys mutation lies between the repressor and homeodomains but the mutant protein was shown in vitro to be unable to bind to its cognate sequences. A novel homozygous missense mutation (126T) of the critical Engrailed homology repressor domain (eh1) of HESX1 was described in a girl born to consanguineous parents (52). Neuroimaging revealed a thin pituitary stalk with anterior pituitary hypoplasia and an ectopic posterior pituitary. Unlike previous cases, she did not have midline or optic nerve abnormalities. Although 126T mutation did not affect the DNA-binding ability of HESX1, it impaired ability of HESX1 to recruit Groucho-related corepressor, thereby leading to partial loss of repression. Tantalizingly, additional nucleotide variants have been described in individuals with isolated GH deficiency, although it is not convincingly clear that these polymorphisms are actually pathogenic (53, 54).

Figure 6

PROP1 (the Prophet of PIT-1) encodes a transcription factor required for the development of most pituitary cell lines, including the somatotrope (GH secretion), lactotrope (prolactin (PRL) secretion), thyrotrope (TSH secretion), and the gonadotrope (FSH and LH secretion). Mutation of PROP1, therefore, results in the deficiency of GH, TSH, PRL, FSH and LH although some individuals with PROP1 mutations have been described with ACTH deficiency (55). Since PROP1 does not appear to be required for the development of the corticotrope cell line, the etiology of ACTH deficiency is unclear. It appears that the ACTH deficiency here is a consequence of the compensatory pituitary hyperplasia that develops over time. Significantly, the degree of TSH deficiency appears to be quite variable, even within mutation-identical individuals, suggesting that the general phenotype associated with PROP1 mutations is also quite variable. PROP1 is encoded by three exons and is located on 5q. Many mutations have been described in PROP1-all inherited in an autosomal recessive manner. Although several studies suggest that mutation of PROP1 is the most common cause of familial MPHD, but is less common in sporadic cases of MPHD (56). Two recurrent mutations have been described, both involving exonic runs of GA tandem repeats (Figure 7). In both cases, the loss of a tandem unit at either locus results in a frameshift and premature termination, and a protein incapable of transactivation.

Figure 7.

POU1F1 encodes the POU1F1 transcription factor, also known as PIT1, which is required for the development and function of three major cell lines of anterior pituitary: somatotropes, lactotropes and thyrotropes. Various mutations in the gene encoding POU1F1 have been described, resulting in a syndrome of multiple pituitary hormone deficiency involving GH, PRL and TSH hormones. POU1F1 is located on 3p11 and consists of six exons encoding 291 amino acids (Figure 8). Many mutations of POU1F1 have been described; some are inherited as autosomal recessive and some as autosomal dominant. There is a wide variety of clinical presentation in patients with POUF1 mutations. Generally, GH and prolactin deficiencies are seen early in life. However, TSH deficiency can be highly variable with presentation later in childhood or normal T4 secretion can be preserved into the 3rd decade (57, 58). To date, POU1F1 mutations have been described in a total of 46 patients from 34 families originating in 17 different countries (59). Recessive mutations are generally associated with decreased activation, while dominant mutations have been shown to bind but not transactivate - i.e. act as dominant-negative mutations, rather than through haploinsufficiency. One such mutation is the recurrent Arg271Trp (R271W), located in exon 6, which results from a C --> T transition at a CpG dinucleotide, i.e. a region predisposed to spontaneous mutagenesis. Another interesting mutation is the Lys216Glu mutation of exon 5. This mutation is unique in that the mutant transcription factor activates both the GH and PRL promoters at levels greater than wild-type (i.e. acts as a superagonist), but down-regulates its own (i.e. the POU1F1) promoter-leading to decreased expression of PIT1. R271W is the most frequent mutation of POU1F1. A recent report describing a novel mutational hot spot (E230K) in Maltese patients suggests a founder effect (58). The same group reported two additional novel mutations within POU1F1 gene; an insertion of a single base pair (ins778A) and a missense mutation (R172Q) (58).

Figure 8.

SOX3 encodes a single-exon gene SOX3, an HMG box protein, located on the X chromosome (Xq26.3) in all mammals (60). It is believed to be the gene from which SRY, testisdetermining gene evolved (61). Based on sequence homology, SOX, however, is more closely related to SOX1 and SOX2, together comprising the SOXB1 subfamily and are expressed throughout the developing CNS (62, 63). In humans, mutations in the SOX3 gene have been implicated in X-linked hypopituitarism and mental retardation. In a single family, a SOX3 gene mutation was shown in affected males who had mental retardation and short stature due to GH deficiency (64). The mutation was an in-frame duplication of 33 bp encoding for an additional 11-alanine, causing an expansion of a polyalanine tract within SOX3. Recently, other mutations including a submicroscopic duplication of Xq27.1 containing SOX3, a novel 7-alanine expansion within the polyalanine tract, and a novel polymorphysm (A43T) in the SOX3 gene were described in males with hypopituitarism. Phenotypes of these patients include severe short stature, anterior pituitary hypoplasia, and ectopic posterior pituitary, colossal abnormalities, and infundibular hypoplasia. Although duplications of SOX3 have been implicated in the etiology of X-linked hypopituitarism with mental retardation, in at least one study, none of the affected individuals had mental retardation or learning difficulties (65). Taken together, the data suggests that SOX3 has a critical role in the development of the hypothalamic-pituitary axis in humans, and mutations in SOX3 gene are associated with X-linked hypopituitarism but not necessarily mental retardation (65).

ISOLATED GH DEFICIENCY (IGHD)

Abnormalities either in the synthesis or the activity of GH can cause a wide variation in the clinical phenotype of the patient. General clinical features of IGHD deficiency include proportionate growth retardation accompanied by a decreased growth velocity, puppet-like facies, frontal bossing, thin hair, a high-pitched voice, moderate trunk obesity, delayed bone maturation and dentition. Patients with IGHD appear younger than their chronological age. Puberty may be delayed until late teens, but usually fertility is preserved.

To date, four Mendelian types of IGHD have been identified on the basis of the type of defect, mode of inheritance, and degree of deficiency.

1) Type 1 GH deficiency is an autosomal recessively inherited condition, which exists as either a complete, or as a partial loss of GH expression.

a) Type 1a deficiency is characterized by the complete absence of measurable GH. Infants born with a type 1a defect are generally of normal length and weight, suggesting that, in utero, GH is not an essential growth factor (66, 67). Growth immediately after birth and during infancy may also be less dependent on circulating GH levels than during other phases of life. Patients with Type 1a deficiency initially respond to rhGH treatment well. However, about 1/3 of patients develop antibodies to GH which leads to markedly decreased final height as adults (59). The exact prevalence of Type 1a deficiency is not known, and most reported families are consanguineous (59). Mutations in Type 1a have been described in GH1 and GHRHR-including nonsense mutations, microdeletions/frame-shifts, and missense mutations.

b) Type 1b deficiency represents a state of partial-rather than an absolute-deficiency of GH, with measurable (but insufficient) serum GH. Therefore it is milder than Type 1a deficiency. Patients with Type 1b deficiency do not typically present with mid-facial hypoplasia or microphallus. They also have a good response to GH treatment without developing GH antibodies. Most cases of Type 1b GH deficiency are caused by missense and/or splice site mutations in the GH1 and GHRHR genes (68).

2) Type 2 GH deficiency is an autosomal dominantly inherited disorder with reduced secretion of GH. Patients with Type 2 GHD usually do not have any pituitary abnormality (69). However, recently, it has been shown that their pituitary may become small over time (70). They have a good response to GH treatment. This type of GH deficiency is intuitively less clear, since autosomal dominant conditions generally occur as a result of either haploinsufficiency or secondary to dominant-negative activity. Haploinsufficiency, however, has not been demonstrated in the obligate heterozygote carriers of individuals harboring GH1 deletions, and is therefore an unlikely explanation. Dominant-negative activity is usually associated with multimeric proteins, also making this explanation less intuitive. Type 2 GHD appears to be the most common form of IGHD and many mutations have been identified in GH1 including splicing and missense mutations (71-79). Recent studies suggest that GH1 may not be the only gene involved in Type 2 GHD. Screening 30 families with autosomal dominant IGHD did not show any GH1 mutations, raising the possibility of other gene(s) may be involved (80).

3) Type 3 growth hormone deficiency is inherited in an X-linked recessive manner. There are no candidate genes and no compelling explanations for this condition. There are no reported mutations of the GH-1 gene in Type 3 GHD. In addition to short stature, patients may also have agammaglobulinemia (59).

Table 6 summarizes phenotype of mutations involved in human pituitary transcription factors causing IGHD and MPHD and their mode of inheritance.

Table 6. Genotype and Phenotype Correlations in Human Pituitary Transcription Factors
Gene	Phenotype	Mode of Inheritance
AR, autosomal recessive; AD, autosomal dominant
IGHD
GH-1	IGHD type 1a/1b IGHD type 2	AR AD
GHRHR	IGHD type 1b	AR
MPHD
1. LHX3	Deficiencies of GH, TSH, LH, FSH, PRL, rigid neck, small/normal/or enlarged anterior pituitary	AR
2. LHX4	Deficiencies of GH, TSH and ACTH, small anterior pituitary cerebellar and skull defects	AD
3. HESX1	Panhypopituitarism, optic nerve hypoplasia, agenesis of the corpus callosum, ectopic posterior pituitary	AR/AD
4. PROP1	Panypopituitarusm except ACTH deficiency, small/normal/or enlarged anterior pituitary	AR
5. POU1F1 (Pit1)	Deficiencies of GH, TSH, PRL, small or normal anterior pituitary	AR/AD
6. SOX3	Hypopituitarism, mental retardation, learning difficulties, small anterior pituitary, ectopic posterior pituitary	X-linked recessive

HYPOTHALAMIC GH DEFICIENCY

Synthesis and Secretion of GH

GHN is synthesized within the somatotropes of the anterior pituitary gland and is secreted into circulation under tripartite control, stimulated by growth hormone releasing hormone (GHRH) and the Growth Hormone Secretagogue, Ghrelin (GHS) and inhibited by somatostatin (SST) (Figure 9). GHRH, GHS and SST secretion are themselves regulated by numerous central nervous system neurotransmitters (Table 5). GH exerts direct metabolic effects on target tissues and exerts many of its growth effects through IGF1, which is produced by the liver and the target tissues (e.g. bones). Additional regulation of GH secretion is achieved through feedback control by IGF1 and GH at the pituitary and at the hypothalamus.

Figure 9. Hypothalamic-pituitary-peripheral regulation of GH Secretion. SST, somatostatin; GHRH, growth hormone releasing hormone; IGF1, insulin-like growth factor type 1

Table 7. Neurotransmitters and Neuropeptides Regulating GHRH Secretion from Hypothalamus.
Dopamine	Gastrin
GABA	Neurotensin
Substance-P	Calcitonin
TRH	Neuropeptide-Y
Acetylcholine	Vasopressin
VIP	CRHs

TIMING

In addition to the absolute GH levels reached, the timing of the GH pulse is also physiologically important. GH is secreted in episodic pulses throughout the day, and the basal levels of GH are often immeasurably low between these peaks (Figure 10). Figure 10 illustrates normal spontaneous daily GH secretion, while figure 11 represents that of a child with GH deficiency.

Figure 10. The characteristic pulsatile pattern of GH secretion in normal children. Note the maximal GH secretion during the night.

Figure 11. GH secretion in a child with GH deficiency. Note the loss (both qualitative and quantitative) of episodic pulses seen in normal children

Approximately 67% or more of the daily production of GH in children and young adults occurs overnight, and most of that during the early nighttime hours that follow the onset of deep sleep. During puberty, there is an increase in GH pulse amplitude and duration, most likely due to estrogens (81). GH secretion is sexually dimorphic, with females having higher secretory burst mass per peak but no difference in the frequency of peaks, or basal GH release (82). In addition, GH secretion is stimulated by multiple physiologic factors (Table 6). Overweight children, independent of pubertal status, have reduced GH levels mainly due to reduced GH burst mass with no change in frequency (83).

Table 8. Physiologic factors that affect GH secretion.
Factors that stimulate GH secretion	Factors that suppress GH secretion
Exercise	Hypothyroidism
Stress	Obesity
Hypoglycemia	Hyperglycemia
Fasting	High carbohydrate meals
High protein meals	Excess glucocorticoids
Sleep

Growth Hormone Releasing Hormone

GHRH (also known as Somatocrinin) is the hypothalamic-releasing hormone isolated in 1982 (84) believed to be the chief mediator of GH secretion from the somatotrope. GHRH deficiency is thought to be the most common cause of 'acquired' GHRH deficiency, secondary to (even mild) birth trauma. GHRH includes 5 exons, with transcription of (the non-coding) exon 1 differing on a tissue-specific basis (85). The mature GHRH protein contains 44 amino acids, with an amidated carboxy-terminus (Figure 12). Despite this post-translational modification, much of the GH-secreting ability resides in the (original) amino half, allowing the synthesis of shorter peptides retaining efficacy (e.g. 1-29 GHRH). Despite being cloned in 1985 (86), there are no reports of (spontaneous) mutations in humans or in any animal model. Individuals with mutations in GHRH are predicted to have isolated GH deficiency.

Figure 12.

Growth Hormone Releasing Hormone Receptor

GHRHR was cloned in 1992 (87), described as the cause of isolated GH deficiency (IGHD) in the Little strain of dwarf mouse by 1993 (88, 89), mapped in the human by 1994 (90), and demonstrated to be a cause of human GH deficiency in 1996 (68). GHRHR is located on 7p15 (90), comprises 13 exons and encodes a protein of 423 amino acids, belonging to the G-protein coupled, heptahelical transmembrane domain receptors (Figure 13). The initial reports of GHRHR mutations were in geographically isolated (and therefore endogamous) populations in South Asia (68, 91, 92) and later in Brazil (93). In fact, haplotype analysis of the GHRHR locus in three unrelated families from the Indian subcontinent, carrying the identical E72X nonsense mutation in GHRHR indicated that this represents a common ancient founder mutation (94). An independent analysis of patients with familial isolated GH deficiency from non-consanguineous families revealed that the majority of patients carried the identical E72X mutation, suggesting that E72X mutation can be a reasonable candidate for isolated GH deficiency (95). There are now numerous other reports, making GHRHR one of the most commonly mutated genes in IGHD. Roelfsema et al studied two members of a single family with an inactivating mutation of the GHRHR and noted that the 'normal' pattern of spontaneous GH production was preserved, although the absolute quantity of GH secreted was quite low and the approximate entropy significantly elevated (96); supporting the view that the amplitude of a GH pulse is the result of a GHRH burst, while the timing of GH pulses is the result of a somatostatin trough.

Figure 13.

Ghrelin

In 1977 Bowers et al (97) reported on the ability of enkephalins to secrete GH and it was later demonstrated that this secretion was independent of GHRH. This sentinel finding gave rise to a new field of study, that of the growth hormone releasing peptides (GHRP's) or growth hormone secretagogues (GHS's). Twenty-two years later Kojima et al (98) reported the isolation of the endogenous ligand whose actions were mimicked by the enkephalins. They named the hormone Ghrelin, based on the Proto-Indian word for 'grow'. GHRELIN is located on 3q25-26 (99), is processed from preproGhrelin precursor, and is primarily produced by the oxyntic cells of the stomach and to a lesser extent in the arcuate ventro-medial and infundibular nuclei of the hypothalamus (100). Ghrelin also plays a role in regulating food intake. In addition to its GH secreting actions, direct intracerebroventricular injection of Ghrelin in mice has potent orexigenic appetite stimulating action, and this action is mediated by NPY, which antagonizes the actions of Leptin.

Several studies have shown that, on a molar basis, Ghrelin is significantly more potent at inducing GH secretion than GHRH (101). Additionally, many of these studies have shown that Ghrelin and GHRH are synergistic, inducing a substantial GH response when given in combination (102-105). Several studies comparing GHRH and Ghrelin demonstrate that 1 ug/kg GHRH results in a GH peak of approximately 25 ng/ml, 1 ug/kg Ghrelin results in a GH peak of approximately 80 ng/ml GH, but when given together, 1ug/kg of GHRH + 1 ug/kg Ghrelin results in a GH peak of approximately 120 ng/ml (105, 106). When short normal children were compared to children with neurosecretory GH deficiency, Ghrelin secretion was similar in both groups during daytime but higher Ghrelin levels were detected during the night in short children with neurosecretory GH deficiency. The authors therefore suggest that Ghrelin is not involved in nighttime GH secretion (107), although these findings are also consistent with a relative Ghrelin insensitivity at night. In a group of boys with constitutional delay of puberty, testosterone administration caused the expected increase in GH concentrations but did not affect the 24-hour Ghrelin profile, suggesting that the testosterone-induced GH secretion was not caused by ghrelin (108). Another study demonstrated a decrease in Ghrelin concentrations following glucagon administration in a group of non-GH-deficient short children, suggesting that Ghrelin does not mediate glucagon-induced GH secretion (109).

A second hormone, Obestatin is also known to be produced from preproGhrelin. Obestatin has anorexigenic effects, opposite those of Ghrelin (110) Several nucleotide changes have been identified in the PREPROGHRELIN locus, and some are associated with body mass index, BMI (111). It is not clear, however, whether these are polymorphisms, or distinct mutations. It is also not clear whether these nucleotide variants exert their effects solely via an altered Ghrelin, a corrupted Obestatin, or a combination of the two. A knockout mouse lacking the preproghrelin locus had no statural or weight phenotype, but this may well be the result of the simultaneous loss of both ghrelin and obestatin. To this point, a transgenic mouse with abnormal ghrelin but normal obestatin did indeed have poor weight gain, explained by either ghrelin deficiency, unopposed obestatin, or both. There are no reports of mutations in GHRELIN associated with short stature, either in humans or in any animal model. The theoretical phenotype of such an individual would presumably be that of isolated GH deficiency, most likely of post-natal onset and possibly with an abnormally low appetite.

Figure 14.

Ghrelin Receptor

The receptor for Ghrelin (GHSR) was identified in 1996 by Howard et al (100)-prior to the identification of the ligand, and maps to 3p26-25. A recent report of a nucleotide variant segregating with short stature in two unrelated families appears to be the first report of a mutated GHSR gene.  The mutation results in a protein with decreased cell-surface _expression, and a resultant decrease in the constitutive activity of the receptor (112).

Combining data from numerous investigators, there appear to be differences in the specific roles of these parallel but independent pathways for GH secretion. Given that:

1.     Ghrelin induces a larger release of GH than GHRH,

2.     Both bolus and continuous GHRH infusion results in a chronic release of GH (113),

3.     A bolus of Ghrelin results in GH secretion, but continuous Ghrelin infusion does not; and

4.     Ghrelin administration (bolus or continuous) does not cause an increase in GH mRNA;

It is therefore likely that the GHRH/GHRHR arm of the somatotropin pathway serves primarily in the production of de novo GH, and secondarily in the release of (pre-made) GH while Ghrelin/GHSR may serve primarily in the release of stored GH, and only secondarily-if at all-in the production of de novo GH (106, 114).

Figure 15.

Somatostatin

The somatostatin gene (SST) is located on 3q28, and contains two exons, encoding a 116 amino acid pre-prosomatostatin molecule that is refined down to a 14 amino acid cyclic peptide (as well as a 28 amino acid precursor/isoform) (115) (see figure 16). Pancreatic somatostatin inhibits the release of both insulin and glucagon, while in the CNS somatostatin inhibits the actions of several hypothalamic hormones, including GHRH. For this reason somatostatin is also known as Growth Hormone Release Inhibiting Hormone. Somatostatin's widespread effects are mediated by five different receptors, all encoded by different genes (rather than through alternative splicing of a single gene). The anti-GHRH actions on the pituitary are primarily mediated by somatostatin receptors (SSTR) 2 and 5, which act by inhibiting cAMP as well as other pathways (116) (see figures 17 and 18). There is a single case report of a nucleotide variant in the SSTR5 gene, occurring in a subject with acromegaly. (This individual, however, was also reported as having a mutation in the GSP oncogene; placing the pathological nature of the SSTR5 variant in question). Whereas GHRH induces release of growth hormone stored in secretory vesicles by depolarization of the somatotrope, somatostatin inhibits GH release by hyperpolarizing the somatotrope, rendering it unresponsive to GHRH. There are no reports of mutations in the somatostatin gene, or in the SSTR2 receptor.

All three of these hypothalamic modifiers of GH secretion act through cell-membrane receptors of the G-protein coupled receptor (GCPR) class. These receptors are characterized by seven membrane-spanning helical domains, an extracellular region that binds (but does not internalize) the ligand hormone, and an intracellular domain that interacts with a G-protein, which contains a catalytic subunit that generates a second messenger (e.g. cyclic AMP or inositol triphosphate).

Figure 16.

Figure 17.

Figure 18.

PITUITARY GH DEFICIENCY

Human Growth Hormone

GH is critical for growth through (most of) childhood as well as for optimal metabolic, neurocognitive, cardiac, musculoskeletal and adipose function throughout life. GH acts through GH receptors on cells of a variety of target tissues. Many, but not all, actions of GH are mediated by insulin-like growth factor 1 (IGF1), also known as Somatomedin-C. IGF1 is released in response to GH and acts as both a hormone and an autocrine/paracrine factor. GH, directly and indirectly through the actions of IGF1, stimulates tissue growth and proliferation, most notably in the epiphyseal growth plates of children, increases lean muscle mass, decreases fat mass, and increases bone mineral density.

Growth hormone is a single-chain polypeptide that contains 191 amino acids with two intramolecular disulfide bonds and the molecular weight of 22,128 Daltons. The GH protein (GHN) is encoded by the GH1 gene located on chromosome 17q22-q24 (Figure 19) in a complex of five genes: two for the growth hormone/growth hormone variant (GH1, GH2), two for chorionic somatomammotropin (CS1, CS2), and one for the somatomammotropin pseudogene (CSL). GH2 encodes the GHV protein that is secreted by the placenta into maternal circulation. GHV has greater lactogenic properties than does GHN, and may function to maintain the maternal blood sugar in a desirable range, thus ensuring sufficient nutrition for the fetus.

Figure 19.

PERIPHERAL GH RESISTANCE

Growth Hormone Receptor

Growth failure with normal serum GH levels is well known, both at the genetic and the clinical level. Although such cases may be due to defects of GH1 (e.g. bioinactive GH), many such subjects have been shown to have mutations in the GH Receptor (GHR), i.e. Growth Hormone Insensitivity, known as Laron Syndrome. Biochemical hallmarks of this syndrome are increased or normal GH levels with low IGF1 and with absent or decreased response to GH treatment (117).

The growth hormone receptor gene (GHR) is located on 5p13-12 and contains 10 exons which span a physical distance of almost 300 kb of genomic DNA. The GHR consists of a ligand-binding extracellular domain, an 'anchoring' transmembrane domain and an intracellular domain with intrinsic tyrosine kinase activity. A monomeric GHR binds a single GH molecule, which then dimerizes a second GHR, and activates the JAK/STAT and MAPK pathways and is internalized. The internalization leads to extinguishing of the GH signal, and the GHR is recycled for further rounds of activity. Two naturally occurring isoforms of the GHR arise from alternative splicing-one with an alternate exon 3, and the other with an alternate exon 9. The alternative exon 9 isoform yields a protein with only amino acids 1-279, and virtually none of the intracellular domain. This isoform cannot transduce the GH signal and yields higher molar quantities of GHBP (than wild-type GHR), and therefore acts as a GH "sink" (118). The GHR isoform lacking exon 3 has a high prevalence, and may be associated with altered GH signaling, although the direction of the alteration is not clear (119-122).

Defects in the GH signaling pathway have been demonstrated to be associated with postnatal growth failure. Mutations of Stat5b were reported in patients with severe growth failure. To date, a total of seven cases with 5 different mutations of Stat5b gene were reported. Although patients had a phenotype similar to that of congenital GH deficiency or GHR dysfunction, clinical and biochemical features (including normal serum GHBP concentrations) distinguish patients with STAT5b defects from patients with GHR defects,. In addition, patients with STAT5b mutations have immunodeficiency defects (163).

In humans, the extracellular portion of GHR is enzymatically cleaved and functions as the GH-Binding Protein (GHBP) (123). GHBP presumably serves to maintain GH in an inactive form in the circulation and to prolong the half-life of GH. Serum levels of GHBP are therefore used as a surrogate marker for the presence of GHR, and abnormal levels-both elevated and decreased-may indicate abnormality in the GHR (124, 125). Of note is that mutations have also been described in individuals with 'normal' GHBP levels. Numerous mutations have been identified in the GHR, most acting as autosomal recessive mutations, but dominant negative mutations have also been described. Individuals with heterozygote mutations in GHR may present with significant short stature (126). Mutations in GHR have also been associated with idiopathic short stature (ISS) (21, 127, 128). The original reports of GHR mutation described limited elbow extension and blue sclera, but are not universal.

Many genetic abnormalities have been described in GHR, including nonsense mutations, missense mutations, macrodeletions, microdeletions and splice site changes. Of the latter, one of the most interesting is the "E180E" mutation, wherein an exonic adenosine is converted to a guanine, converting GAA --> GAG, which is predicted to encode glutamic acid and glutamic acid; that is to not alter the coding of the GHR. On this basis, this "silent polymorphism" would be expected to have no phenotype, but instead causes GH resistance and extreme short stature by activation of a cryptic splice site. This mutation was noted in Loja and El Oro, Ecuador in two large cohorts. This identical mutation has also been identified in Jews of Moroccan descent, suggesting that this mutation dates back to at least the 1400s and that the Ecuadorian cohorts, therefore, represent Sephardic Jews who left Spain around the time of the Inquisition at the end of the fifteenth century (129, 130). Another splice site mutation at position 785-3 (C>A in the intron 7) was recently described in a patient and mother with short stature and extremely elevated GHBP (164). The consequence of this novel mutation is a truncated GHR which lacks the transmembrane domain (encoded by exon 8) and the cytoplasmic domain. It was hypothesized by the authors that this GHR variant cannot attach to the cell membrane, and the continual secretion into the circulation results in the elevated levels of serum GHBP detected in the patient and his mother. The presence of the wild-type GHR allele presumably permits some level of normal GH-induced action.

Figure 20

Insulin-Like Growth Factor 1 (IGF1)

Many of growth hormone's physiologic actions are mediated through the insulin-like growth factor, IGF1 (formerly referred to as somatomedin C). Serum IGF1 levels are commonly measured as a surrogate marker of GH status, since IGF1 displays minimal circadian fluctuation in serum concentration. IGF1 plays a critical role in both prenatal and postnatal growth, signaling through the IGF1 as well as the insulin receptor. IGF1 circulates as a ternary complex consisting of IGF1, IGBP3 and ALS. The IGF1 gene is located on 12q22-24.1, consists of six exons and spans over 45 kb of genomic DNA. Alternative splicing produces two distinct IGF1 transcripts, IGF1-A and IGF1-B. Woods et al described a male of a consanguineous union with prenatal (intrauterine) and postnatal growth retardation, sensorineural deafness and mental retardation (131). DNA analysis showed a homozygous partial deletion of the IGF1 gene (131). Subsequently, additional cases have been described (132, 133).

Mice engineered to completely lack Igf1 (Igf1 knockouts) are born 40% smaller than their normal littermates (134, 135). Recent studies of a hepatic-only Igf1 knockout (KO) mouse, however, demonstrate that IGF1 functions primarily in a paracrine or autocrine role, rather than in an endocrine role (136). Liver specific Igf1 knock-out mice, were found to have a 75% reduction in serum Igf1 levels but were able to grow and develop (nearly) normally (136, 137) with a mild phenotype developing only late in life (136). A further decrease in serum IGF1 levels of 85% was observed when double gene KO mice were generated lacking both the acid labile subunit (ALS) and hepatic IGF1. Unlike the single hepatic-only IGF1-KO's, these mice showed significant reduction in linear growth as well as 10% decrease in bone mineral density (138). Thus, as illustrated by the combination liver specific IGF1+ALS knock-out mouse model, there likely exists a threshold concentration of circulating IGF1 that is necessary for normal bone growth and suggests that IGF1, IGFBP3, and ALS may play an important role in bone physiology and the pathophysiology of osteoporosis.

In humans, homozygous mutations in ALS result in mild postnatal growth retardation, insulin resistance, pubertal delay, unresponsiveness to GH stimulation tests, elevated basal GH levels, low IGF1 and IGFBP3 levels and undetectable ALS (165-167). Although it is not clear why postnatal growth is mildly affected, it might be due to increased GH secretion due to loss of negative feedback regulation by the low circulating IGF1. Increased GH secretion could then up-regulate the functional GH receptor increasing local IGF1 production, thus protecting linear growth (Figure 21) (168).

Figure 21. Savage MO Camacho-Hubner C, David A, et al. 2007 Idiopathic short stature: will genetics influence the choice between GH and IGF1 therapy? Eu J of Endocrine 157:S33 Society of European Jouirnal of Endocrinology (2007). Reproduced by permission. Reprinted with permission (168).

Elevated IGF1 levels has recently been associated with colon, prostate and breast cancer (139-141) and the association was strongest when an elevated IGF1 was combined with a decreased IGFBP3 level. This combination-expected to yield more bioactive IGF1-may merely reflect the tumorigenic process, rather than demonstrate causality. Importantly, GH treatment induces a rise in both IGF1 as well as IGFBP3 (142), and therefore would not be expected to increase cancer risk in normal individuals.

Table 9. Summary of IGF1 function in different systems and its effects: (169).

Table 9. Summary of IGF1 function in different systems and its effects: (169).
IGF1 Function	IGF1 Deficiency
Intrauterine Growth	IUGR
Postnatal Growth	Short Stature
CNS	Neurodegenerative disease
Insulin sensitization/improvement of glucose disposal/beta cell proliferation	Type 1 and Type 2 Diabetes
	IGF1 Excess
Mitosis/Inhibition of apoptosis	Malignancy

Figure 22

IGF1 Deficiency

IGF1 deficiency can be classified based on decreased IGF1 synthesis (primary) or decreased IGF1 secondary to decreased or inactive GH (secondary) (170). This doesnt really differentiate between 1 and 2 both have decreased synthesis. The only good way that I can think of is associated with high or low GH, as below

1. Primary IGF1 Deficiency (normal or elevated GH levels):
1. Defects in IGF1 production
1. Mutation in IGF1 gene or bioinactive IGF1
2. GHR receptor signaling defects (JAK/STAT)
3. Mutations in ALS gene
4. Factors effecting IGF1 production (malnutrition, liver disease)
2. Defects in IGF1 action
1. IGF1 resistance due to receptor or post-receptor defects
2. Factors inhibiting IGF1 binding to IGF1R (increased IGFBPs and presence of IGF1 antibodies)
3. Defects in GH action
1. Factors inhibiting GH binding to GHR (increased GHBPs and presence of GH antibodies)
2. GH receptor defects (decreased GH receptors, GHR antibodies, GHR gene defects)
2. Secondary IGF1 Deficiency (decreased GH levels):
1. Decreased GH production
1. Defects in GH gene
2. Defects in GHRH or GHRH receptor
3. Neocortical/psychological
2. Defects in hypothalamus and pituitary

The clinical features of primary IGF1 deficiency are similar to severe GH deficiency.

Recombinant human IGF1 (rhIGF1)

rhIGF1 is useful in the treatment of primary IGF1 deficiency resulting from abnormalities of the GH molecule (resulting in a bioinactive GH), the GH receptor (known as Laron syndrome) or GH signaling cascade (39). Studies have shown that rhIGF1 significantly improves height in children unresponsive to rhGH (40, 41), and clinical trials clearly demonstrated better response to IGF1 therapy when initiated at an early age (42).

FDA approved conditions for rhIGF1 treatment for children includes (171):

1.     Severe primary IGF1 deficiency

2.     GH gene deletions who have developed neutralizing antibodies to GH

Severe primary IGF1 deficiency is defined by:

1.     Height SD score is less than -3SD

2.     Basal IGF1 level is below -3SD

3.     Normal or elevated GH

The recommended usual dose in children is 100-200 microgram/kg/day (43).

The most common side effects of IGF1 treatment are pain at injection site and headaches which mostly diminishes after first month of treatment (39). Other less common side effects are lipohypertrophy at the injection site, pseudotumor cerebri, facial nerve palsy and hypoglycemia (42). Another effect of IGF1 treatment is a significant increase in fat mass and BMI (44)in contradistinction to the lipolytic effect of rhGH treatment. Coarsening of facial feature and increased hair growth are other prominent side effects and most commonly are seen during puberty. Growth of lymphoid tissue is a concern which may require tonsillectomy (39).  

Insulin-Like Growth Factor 1 Receptor (IGF1R)

The receptor for IGF1 is structurally related to the insulin receptor and similarly has tyrosine kinase activity (Figure 23). IGF1R is located on 15q25-26. The mature (human) IGF1 receptor contains 1337 amino acids and has potent anti-apoptotic activity (143). The IGF1 receptor transduces signals from IGF1, IGF2 and insulin. However, murine data suggest that initially (in the fetus) only the IGF2 signal is operational, while later on in development, both IGF1 and IGF2 (and probably insulin) signal through the IGF1R (144). Hemizygosity for IGF1R has been reported in a single patient (and appears likely in seven others) with IUGR, microcephaly, micrognathia, renal anomalies, lung hypoplasia and delayed growth and development (145). Murine and human studies have shown that mutations in IGF1R result in combined intrauterine and postnatal growth failure (146), confirming the critical role of the IGF system on embryonic, fetal and postnatal growth. A novel heterozygous mutation in the tyrosine kinase domain of the IGF1R gene was recently identified in a family with short stature. The mutation, a heterozygous 19-nucleotide duplication within exon 18 of the IGF1R gene, results in a haploinsuffiency of IGF1R protein due to nonsense mediated mRNA decay (172).

Figure 23.

Insulin-Like Growth Factor 2

IGF2 is thought to be a major prenatal growth hormone and less important in post-natal life.

The human gene, IGF2, is located on 11p15.5. Chromosome 11p15.5 carries a group of maternally (IGF2) and paternally (H19) imprinted genes that crucial for the fetal growth. Genetic or epigenetic changes in the 11p15.5 region alter the growth (147). IGF2 is maternally imprinted, meaning that the maternal allele is unexpressed. The close proximity of the INS to IGF2-in addition to nearly 50% amino acid identity-suggest that these genes arose through gene duplication events from a common ancestor gene. IGF2 acts via the IGF1 receptor (as well as the insulin receptor). Over-expression of IGF2 results in overgrowth, similar to that seen in Beckwith-Wiedemann Syndrome (which can be due to loss of imprinting, effectively doubling IGF2 expression). A mouse model overexpressing Igf2 demonstrates increased body size, organomegaly, an omphalocele, cardiac, adrenal and skeletal abnormalities, suggestive of Beckwith-Wiedemann and Simpson-Golabi-Behmel syndromes (148). Interestingly, IGF2 expression is normally extinguished by the Wilm's Tumor protein (WT1), providing an explanation for the overgrowth (e.g. hemi-hypertrophy) typically seen in subjects with Wilm's Tumor (149). In contrast, mice without a functional Igf2 (Igf2 knockouts) are born 40% smaller than their normal littermates (identical to Igf1 knockouts).

Recent reports on individuals with severe intrauterine growth retardation showed maternal duplication of 11p15 (150). Furthermore, individuals with Russell-Silver syndrome (SRS, also known as Russell-Silver syndrome) have been found to have an epimutation (demethylation) associated with biallelic expression of H19 and down regulation of IGF2 (151, 152). Russell-Silver syndrome is a congenital disorder characterized by intrauterine and postnatal growth retardation, typical facial features (triangular face, micrognathia, frontal bossing, downward slanting of corners of the mouth), asymmetry, and clinodactyly. Other chromosomal abnormalities such as maternal uniparental disomy on chromosome 7 also have been shown in 10% of individuals with SRS (153).

A paternally derived balanced chromosomal translocation that disrupted the regulatory regions of the predominantly paternally expressed IGF2 gene was described in a woman with short stature, history of severe intrauterine growth retardation (-5.4 SDS), atypical diabetes and lactation failure (173).

Figure 24.

Insulin-Like Growth Factor 2 Receptor

A receptor for IGF2, IGF2R, has been identified, but does not appear to be the mediator of IGF2's growth promoting action. IGF2R is located on 6q26, and encodes a receptor unrelated to the IGF1 or insulin receptor. IGF2R is also the mannose-6-phosphate receptor and serves as a negative modulator of growth (for all IGF's and also insulin). Its main role in vivo is probably as a tumor suppressor gene. While IGF2 is maternally imprinted, mouse Igf2R is paternally imprinted. There is some evidence that (in a temporally-limited fashion) IGF2R is also paternally imprinted in humans. Somatic mutations have been found in hepatocellular carcinoma tissue (heterozygous mutations associated with loss of the other allele), but no germ-line mutations have been identified in individuals with growth abnormalities.

Figure 25.

Insulin

In addition to its glycemic and metabolic roles, insulin functions as a significant growth promoting/anabolic agent. The insulin gene (INS) is located on Chr 11p15.5 and comprises 3 exons. Insulin's role in fetal growth is quite significant, as hyperinsulinemic babies (e.g. infants of diabetic mothers (IDM)) clearly demonstrate. Insulin's growth promoting activity is mediated through a combination of the insulin and the IGF1 receptors. Mutations in the INS gene have been described in subjects with hyperinsulinemia (and/or hyperproinsulinemia) and diabetes mellitus.

Figure 26.

Insulin Receptor

The insulin receptor is structurally related to the IGF1 receptor. The gene, INSR, is located on Chr 19p13.2 and contains 22 exons that span over 120 kilobases of genomic DNA. INSR encodes a transmembrane protein with tyrosine kinase activity which is capable of transducing the signals of insulin, IGF1 and IGF2.

Individuals with a mutation in the insulin receptor have been identified and appear to be the basis for the mythological 'Leprechauns'. They typically have intrauterine growth retardation; small elfin facies with protuberant ears; distended abdomen; relatively large hands, feet, and genitalia; and abnormal skin with hypertrichosis, acanthosis nigricans, and decreased subcutaneous fat. At autopsy, several subjects have been found to have cystic changes in the membranes of gonads and hyperplasia of pancreatic islet cells. Severe mutations generally lead to death within months, but more mild mutations have been found in individuals with insulin resistance, hypoglycemia, acanthosis nigricans, normal subcutaneous tissue and may even be associated with a normal growth pattern! Individuals with even 'mild mutations' have been shown to have a thickened myocardium, enlarged kidneys and ovarian enlargement.

Figure 27.

THE SHORT STATURE homeobox-containing gene (SHOX) HAPLOINSUFFICIENCY

Figure 28. Reprinted with Permission (174).

The Short Stature Homeobox-containing Gene (SHOX) was identified in the pseudoautosomal region 1 on the distal end of the X and Y chromosomes at Xp22.3 and Yp11.3 (Figure 28)(175). Mutations in SHOX were observed in 60-100% of Lri-Weill dyschondrosteosis and Langer mesomelic dysplasia (176, 177). Today it is estimated that SHOX mutations occur with an incidence of roughly 1:1,000 newborns, making mutations of this gene one of the most common genetic defects leading to growth failure in humans.

Genes in pseudoautosomal region 1 do not undergo X inactivation, therefore, healthy individuals express two copies of the SHOX gene, one from each of the sex chromosomes in both 46,XX and 46,XY individuals. The SHOX gene plays an important role in linear growth and is involved in the following:

1. intrauterine linear skeletal growth

2. Fetal and childhood growth plate in a developmentally specific pattern and responsible for chondrocyte differentiation and proliferation (178).

3. A dose effect: SHOX haploinsuffiency is associated with short stature. In contrast, SHOX overdose as seen in sex chromosome polyploidy is associated with tall stature.

More than 50 different mutations (including deletions) of the SHOX gene have been described (174). SHOX abnormalities demonstrate a broad phenotypic spectrum, ranging from short stature without dysmorphic signs as seen in idiopathic short stature (ISS) to profound Langers mesomelic skeletal dysplasia, a form of short stature characterized by disproportionate shortening of the middle segments of the upper arms (ulna) and lower legs (fibula) (179).

Rappold et al. developed a scoring system to determine the phenotypic spectrum of SHOX deficiency in children with short stature and identify patients for SHOX molecular testing (180). The authors recommend a careful examination including measurement of body proportions and X-ray of the lower legs and forearm before making the diagnosis of ISS. The scoring system consists of three anthropometric variables (arm span/height ratio, sitting height/height ratio and BMI), and five clinical variables (cubitus valgus, short forearm, bowing of forearm, muscular hypertrophy and dislocation of the ulna at the elbow). Based on the scoring system, authors recommend testing for SHOX deficiency for the individuals with a score greater than four or seven out of a total score of 24 (Table 10).

The recent data show that GH treatment is effective in improving linear growth of patients with SHOX mutations (179).

Table 10. Scoring system for identifying patients that qualify for short-stature homeobox containing gene (SHOX) testing based on clinical criteria. Reprinted with permission (180).
Score item	Criterion	Score points
Arm span/height ratio	<96.5%	2
Sitting height/height ratio	>55.5%	2
Bodymass index	>50th percentile	4
Cubitus valgus	Yes	2
Short forearm	Yes	3
Bowing of forearm	Yes	3
Appearance of muscular hypertrophy	Yes	3
Dislocation of ulna (at elbow)	Yes	5
Total		24

NOONAN SYNDROME (NS)

Noonan syndrome (NS) is a relatively common genetic disorder with the incidence of between 1:1000 and 1:4000 (181). NS is inherited in an autosomal dominant manner, and sporadic cases are not uncommon (50-60%) (182). NS is characterized by short stature, cardiac defects (most commonly pulmonary stenosis and hypertrophic cardiomyopathy), facial dysmorphism (down-slanting, antimongoloid palpebral fissures, ptosis, and low-set posteriorly rotated ears), webbed neck, mild mental retardation, cryptorchidism, feeding difficulties in infancy. The phenotype is variable between affected members of the same family and becomes milder with age (183).

Nearly 50% of patients with NS have gain-of-function mutations in PTPN11, the gene encoding the protein tyrosine phosphatase SHP-2 (184). Mutations in four other genes (KRAS, SOS1, NF1 and RAF1) involved in RAS/MAPK signaling systems have been identified in patients with the NS phenotype and related disorders including LEOPORD, Costello, and cardio-facial-cutaneous syndromes (Figure 29) (185).

Figure 29. Reprinted with permission (185)

Although identifying these mutations has contributed to better understanding of the pathogenesis of NS, it appears that the genotype does not completely correlate with the phenotype, e.g. short stature in patients with NS. Several studies have shown that the PTPN11 mutation tended to be associated with poor growth and resistance to GH (186,187). However, data from one large study of individuals with NS did not demonstrate the same correlation between PTPN11 mutations and short stature (181). Albeit, more recent studies showed significant improvement in final adult height in individuals with NS regardless of their mutation type (188,189).

CONCLUSION

The genetic control of human growth is becoming increasingly clear. Many genes have been identified that contribute to the development and function of the pituitary gland including the somatotrope and the GH/IGF1 axis. Defects in many of these genes have already been shown to be responsible for abnormal growth in humans. However, elucidation of these genetic factors provides us with a better understanding of the physiology of growth, and should lead to the improved diagnosis and treatment of individuals with growth abnormalities.