The Peptide Receptor Group

The Glycoprotein Hormone Receptor Group

The glycoprotein hormones include TSH, FSH, LH, and HCG. These hormones share common α subunits that dimerize with hormone-specific β subunits. TSH, FSH, and LH bind to the extracellular amino-terminal domain of the TSH, FSH, and LH receptors, respectively. The effects of HCG are mediated by the LH receptor, which is also known as the luteinizing hormone/choriogonadotropin receptor ( LHCGR ).

Glycoprotein hormone receptors have a large (350 to 400 residues) extracellular amino-terminal domain, also known as the ectodomain , that participates in ligand binding (see Fig. 3.3 ). The ectodomain includes leucine-rich repeats that are highly conserved among the glycoprotein hormone receptors. There is 39% to 46% similarity of the ectodomain and 68% to 72% similarity of the transmembrane or serpentine domain among the three glycoprotein hormone receptors.

The glycoprotein hormone receptors are coupled to G _s , and hormone binding stimulates adenylyl cyclase, leading to increased intracellular cyclic AMP levels and protein kinase A (PKA) activation. Mutations leading to endocrine dysfunction have been reported for each of the glycoprotein hormone receptors.

Thyroid-Stimulating Hormone Receptors

The TSH receptor gene is located on chromosome 14 and contains 10 exons, with the first nine exons encoding the large extracellular domain and the 10 ^th exon coding the remainder of the receptor. At low extracellular TSH concentrations, TSH receptor activation leads to stimulation of Gα _s —which activates adenylyl cyclase, resulting in increased intracellular cyclic AMP levels. At higher extracellular TSH concentrations, activation of the TSH receptor also stimulates the G _q and G ₁₁ proteins—activating phospholipase C and resulting in the production of diacylglycerol and inositol phosphate.

TSH receptors differ from the other glycoprotein hormone receptors in that they exist in two equally active forms. These are the single-chain and two-subunit forms of the TSH receptor ( Fig. 3.4 ). The single-chain form of the TSH receptor is made up of three contiguous subunits: the A subunit, C peptide, and B subunit. The A subunit begins at the amino-terminal of the extracellular domain and contains most of the extracellular domain. The C peptide is connected to the carboxy-terminal of the A subunit and continues the extracellular domain. The C peptide contains a 50-amino-acid sequence that is only found in TSH receptors. The B subunit is connected to the C terminal of the C peptide and contains the TMs and the carboxy-terminal cytoplasmic portion of the receptor. The two-subunit form of the receptor is missing the C peptide, which is cleaved from the protein during intracellular processing and consists of the A and B subunits attached by disulfide bonds. It is surprising that both receptor forms are activated equally by TSH because the C peptide and nearby regions of the A and B subunits participate in signal transduction.

The thyroid-stimulating hormone (TSH) receptor. There are two forms of TSH receptors. The single-chain form is made up of an A subunit, C peptide, and B subunit. Posttranslational cleavage of the C peptide from the single chain form results in the two-subunit form. This form consists of the A subunit joined to the B subunit by disulfide bonds between the carboxy-terminal cysteine residues of the A subunit and the amino-terminal cysteine residues of the B subunit.

(From Rapoport, B., Chazenbalk, G. D., Jaume, J. C., McLachlan, S. M. (1998). The thyrotropin (TSH) receptor: interaction with TSH and autoantibodies. Endocr Rev , 19, 676. Copyright 1998, The Endocrine Society. With permission.)

Missense mutations of the TSH receptor gene, leading to replacement of Ser-281 near the carboxy-terminus of the A subunit, with Ile, Thr, or Asn, result in a constitutively active TSH receptor that may cause intrauterine or congenital hyperthyroidism, or toxic adenomas. Activating somatic mutations that cause toxic adenomas have also been found in different transmembrane domains of the TSH receptor. More specifically, clusters of mutations are located in the i3 and TM6 regions—found to be involved with signal transduction in all glycoprotein hormone receptors. The prevalence of activating mutations of the TSH receptor in toxic adenomas has been estimated to range from 2.5% in Japan to 86% in Brazil.

Activating somatic mutations of the TSH receptor have also been found in multinodular goiters. Interestingly, different activating mutations have been found in separate nodules in the same individual. Some well-differentiated thyroid carcinomas have activating mutations of the TSH receptor. Somatic activating mutations of the GNAS gene encoding Gα _s have also been found in some toxic adenomas and differentiated thyroid carcinomas. Activating germline mutations of the TSH receptor can cause sporadic or autosomal dominant inherited nonautoimmune hyperthyroidism that presents in utero, during infancy, during childhood, and in some cases in adulthood. These mutations have been found in the amino-terminal extracellular and transmembrane domains.

Patients with heterozygous mutations that lead to constitutively active TSH receptors typically develop hyperthyroidism. In contrast, biallelic loss of function mutations in the TSH receptor genes cause hypothyroidism. Most known loss-of-function TSH receptor mutations are located in the amino-terminal extracellular domain. A spontaneous p.Asp410Asn substitution, near the carboxy-terminus of the C peptide, results in a TSH receptor, with normal ligand binding affinity and impaired Gα _s -mediated signal transduction. Patients who are homozygous for this mutation present with compensated hypothyroidism.

Patients homozygous or compound heterozygous for loss-of-function mutations of the TSH receptor present with the syndrome of resistance to TSH (RTSH). Loss-of-function mutations of the TSH receptor that cause RTSH have been identified in the amino-terminal extracellular domain, TM4, TM6, i2, e1, and e3. Clinical severity of RTSH may range from a euthyroid state accompanied by elevated TSH levels (fully compensated RTSH), to mild hypothyroidism unaccompanied by a goiter (partially compensated hypothyroidism), to congenital thyroid hypoplasia accompanied by profound hypothyroidism (uncompensated RTSH). In patients with uncompensated RTSH, a small bilobar thyroid gland is located at the normal site. Loss-of-function mutations of the TSH receptor are a rare cause congenital hypothyroidism more common in Japan and Taiwan (≤ 7% of children), where p.R450H is particularly frequent. Because expression of the sodium-iodide symporter is TSH dependent, thyroid gland uptake of iodine and ^99m pertechnetate is diminished or absent in patients with RTSH. In rare cases, iodine uptake is high-normal. These compound heterozygous mutations of the TSH receptor had some Gα _s activity and no G _q activity, suggesting that iodine uptake is solely controlled by Gα _s activity and not G _q activity. Some families have been found to have an autosomal-dominant form of RTSH that is not caused by a mutation of the TSH receptor.

The Gonadotropin-Releasing Hormone Receptor Group

The Thyrotropin-Releasing Hormone and Secretagogue Receptor Group

Other Class A Receptors That Transduce Hormone Action

Growth Hormone–Releasing Hormone Receptor

The GHRH receptor gene is located at 7p14. GHRH receptors interact with G _s to stimulate adenylyl cyclase, resulting in increased intracellular cyclic AMP levels that lead to somatotroph proliferation and GH secretion. Thus it is not surprising that activating mutations in Gα _s leading to constitutive activation of adenylyl cyclase have been found in some GH–secreting pituitary adenomas in humans.

Many mutations in the GHRH receptor that cause isolated GH deficiency have been identified. These include six splice site mutations, two microdeletions, two nonsense mutations, one frameshift mutation, 10 missense mutations, and one mutation in the promoter. The first naturally occurring mutation in the GHRH receptor (p.D60G) was found in the little mouse, which has a dwarf phenotype. This mutation in a conserved amino acid in the extracellular domain impairs the ability to bind GHRH. The first human mutation in the GHRH receptor (p.Glu72X) was identified in a consanguineous Indian family. The same mutation was found in three apparently unrelated consanguineous kindreds from India, Pakistan, and Sri Lanka. A different mutation (5’ splice site mutation in intron 1) was identified in a large Brazilian kindred of more than 100 individuals. Both mutations result in the production of markedly truncated proteins with no receptor activity. A frameshift mutation was identified in a patient with severe short stature and was the first documented case of early-onset anterior pituitary hypoplasia. In another family, two siblings with isolated GH deficiency were found to be compound heterozygous for inactivating GHRH receptor gene mutations. Three more novel mutations were identified in families with severe short stature in the United Kingdom (p.W273S, p.R94L, and p.R162W). The only mutation found in the promoter region of GHRH affects one of the Pit-1 binding sites.

Studies of subjects in these large kindreds who are homozygous or compound heterozygous for inactivating GHRH receptor gene mutations have shown that affected children experience severe postnatal growth failure with proportionate short stature. Males have high-pitched voices and moderately delayed puberty. Unlike infants with complete GH deficiency, they do not have frontal bossing, microphallus, or hypoglycemia. Other features include a doll facies, reduced muscle mass, central adiposity, wrinkled thin skin, and delayed pigmentation of the hair in children and teens. Bone age is delayed with respect to chronologic age, but advanced with respect to height age. Some patients have been found to have pituitary hypoplasia. Growth velocity increased with exogenous GH therapy. Remarkably, GH treatment of two siblings from Turkey, with the p.E72X mutation, allowed them to reach a normal adult height, despite pretreatment heights of − 6.7 and –8.6 SD and initiation of GH around age 14 years.

Further studies in the Brazilian cohort revealed that homozygotes had increased abdominal obesity, a higher low-density lipoprotein (LDL), and total cholesterol but normal carotid wall thickness and no evidence of premature atherosclerosis. Treatment of these patients with GH for 6 months improved body composition, reduced LDL and total cholesterol, and increased high-density lipoprotein (HDL). Surprisingly, this was associated with increased carotid intima-media thickness and atherosclerotic plaques. Reevaluation, 5 years after the discontinuation of GH, showed a return to baseline for these measures. Patients with a null mutation affecting the GHRH receptor also exhibited altered sleep patterns with abnormalities in both rapid eye movement and nonrapid eye movement sleep. Heterozygotes for the null mutation had normal adult heights and IGF-1 SD scores but exhibited reduced body weight, BMI, lean mass, fat mass, and increased insulin sensitivity.

SNPs in the GHRH receptor have been shown to contribute to height-SDS variation, but mutations remain a rare cause of isolated GH deficiency.

Gastric Inhibitory Polypeptide Receptors

The gastric inhibitory peptide receptor ( GIPR ) gene is located on the long arm of chromosome 19. Two functional isoforms exist in humans because of alternate splicing. GIPR activation induces Gα _s activation of adenylyl cyclase. Gastric inhibitory polypeptide (GIP) is also known as glucose-dependent insulinotropic polypeptide and is released by K cells in the small intestine in response to food. GIP has numerous physiologic actions, including stimulation of glucagon, somatostatin, and insulin release by pancreatic islet cells. Human mutations in the GIPR have not been identified to date. One study identified an SNP in the GIPR that is associated with insulin resistance in obese German children. Another study identified an SNP in the GIPR that is associated with reduced fasting and induced C-peptide levels.

The GIPR is implicated in food-dependent Cushing syndrome. Circulating cortisol levels in patients with food-dependent or GIP-dependent Cushing syndrome rise abnormally in response to food intake. GIP does not normally induce cortisol release from adrenocortical cells. These patients may have adrenal adenomas or nodular bilateral adrenal hyperplasia that overexpresses GIPRs that abnormally stimulate cortisol secretion when activated. Thus in these patients postprandial GIP release leads to activation of these abnormally expressed and functioning adrenal GIPRs, resulting in excessive adrenal cortisol secretion.

Parathyroid Hormone and Parathyroid Hormone–Related Peptide Receptors

Two types of PTH receptors have been identified. The type 1 PTH receptor (PTHR1) is activated by PTH and parathyroid hormone–related peptide (PTHrP) and mediates PTH effects in bone and kidney. In spite of 51% homology to the PTHR1, the type 2 PTH receptor (PTHR2) is activated by PTH but not PTHrP. The PTHR2 is particularly abundant in the brain and pancreas but is also expressed in the growth plate; its natural ligand is TIP39. The function of the PTHR2 is largely unknown.

The PTHR1 has a large amino-terminal extracellular domain containing six conserved cysteine residues. Ligand binding induces the PTHR1 to interact with G _s and G _q proteins, leading to activation of the adenylyl cyclase/PKA and phospholipase C/protein kinase C second-messenger pathways, respectively. Interestingly, mutations in i2 interfere with coupling of the PTHR1 to G _q , without interfering with coupling to G _s —whereas mutations in i3 disrupt coupling of the receptor to both G proteins. Binding of PTH to the PTHR1 leads to internalization of a portion of plasma membrane containing a ternary complex of activated receptor-Gsα-adenylyl cyclase that exhibits sustained production of cyclic AMP. In contrast, PTHrP binding to the PTHR1 receptor leads to formation of a complex that remains on the cell surface and generates cyclic AMP for only a short period of time.

Biallelic loss-of-function mutations in the PTHR1 gene cause Blomstrand chondrodysplasia. This lethal disorder is characterized by accelerated chondrocyte differentiation, resulting in short-limbed dwarfism, mandibular hypoplasia, lack of breast and nipple development, and severely impacted teeth. One patient with this rare condition was found to be homozygous for a point mutation that resulted in a p.Pro132Leu substitution in the amino-terminal domain that interferes with ligand binding. Another patient was found to be homozygous for a frameshift mutation that results in a truncated receptor lacking TM5-7, and contiguous intracellular and extracellular domains.

A third patient was found to have a maternally inherited mutation that altered splicing of maternal mRNA, resulting in a PTHR1 with a deletion of residues 373 through 383 in TM5 (which also interferes with ligand binding). In spite of heterozygosity for the mutation, the patient was unable to produce normal PTHR1s, because for unknown reasons the paternal allele was not expressed. Heterozygosity for somatic or germline p.Arg150Cys missense mutations in the PTHR1 was identified in two out of six patients with enchondromatosis, a condition that is usually sporadic and which is attributed to a postzygotic somatic cell mutation. Enchondromas are benign cartilage tumors that develop in the metaphyses and may become incorporated into the diaphyses of long tubular bones, in close proximity to growth plate cartilage; there is an increased risk of malignant transformation to osteosarcoma. Patients with multiple enchondromatosis (OMIM ID: 166000) have Ollier disease (World Health Organization terminology), a disorder characterized by the presence multiple enchondromas with an asymmetric distribution of lesions that vary in size, number, and location. When multiple enchondromatosis occurs with soft tissue hemangiomas, the disorder is known as Maffucci syndrome . In vitro studies showed that the p.Arg150Cys mutation was mildly activating but led to stimulation of phospholipase C rather than adenylyl cyclase. A transgenic knockin mouse expressing the mutant PTHR1, under the control of the collagen type 2 promoter, showed development of tumors that are similar to those observed in human enchondromatosis. The clinical significance of these observations are uncertain, as most cases of enchondromatosis are caused by mutations in the IDH1 and IDH2 genes.

Biallelic loss of function mutations in PTHR1 are also the cause for Eiken syndrome, which is characterized by a skeletal dysplasia with severely retarded ossification, principally of the epiphyses, pelvis, hands, and feet, as well as abnormal modeling of the bones. Duchatelet et al. mapped Eiken syndrome to chromosome 3p near the PTHR1 gene. Affected individuals were homozygous for a nonsense mutation in the carboxy-terminal cytoplasmic tail of the PTHR1 gene (p.R485X). In a 7-year-old boy with Eiken syndrome, who was born to unaffected first-cousin parents, Moirangthem et al. identified a homozygous missense mutation at a conserved residue in the PTHR1 gene (p.E35K). Finally, nonsyndromic primary failure of tooth eruption (PFE) is caused by heterozygous mutations in the PTHR1 gene. Three distinct mutations, namely c.1050-3C > G, c.543 + 1G > A, and c.463G > T, were identified in 15 affected individuals from four multiplex pedigrees. All mutations truncate the mature protein and therefore should lead to a functionless receptor.

Some cases of Jansen metaphyseal chondrodysplasia have been found to be caused by constitutively activating mutations of the PTHR1 gene. This autosomal-dominant disorder is characterized by short-limbed dwarfism caused by impaired terminal chondrocyte differentiation and delayed mineralization, accompanied by hypercalcemia. Interestingly, constitutive activation appears to result predominantly in excessive Gα _s activity because adenylyl cyclase activity is increased and phospholipase C activity is unchanged in COS-7 cells expressing mutated receptors.

Other Class B Receptors That Transduce Hormone Action

Other class B receptors that transduce hormone action include glucagon-like peptide-1, glucagon, calcitonin, and corticotrophin-releasing factor receptors. Class B receptors usually couple with heterotrimeric G _s proteins, leading to the activation of adenylyl cyclase—which in turn leads to elevated intracellular cyclic AMP levels. (See chapter on Diabetes Mellitus. for a discussion of the role of GLP1 in promoting insulin secretion and the use of GLP1 analogues or inhibitors of GLP1 breakdown in therapy.)

Calcium-Sensing Receptors

The calcium-sensing receptor (CaSR) is located on the long arm of chromosome 3 (3q21.1). The CaSR has a large amino-terminal domain that contains nine potential glycosylation sites. Binding of ionized calcium to the CaSR leads to activation of phospholipase C via activation of G _q/11 proteins.

The CaSR is an integral component of a feedback system that uses PTH and renal tubular calcium reabsorption to keep the serum concentrations of ionized calcium within a narrow physiologic range. Increased extracellular ionized calcium concentrations activate CaSRs in parathyroid chief and renal tubular epithelial cells, leading to decreased PTH release and renal tubular calcium reabsorption. When ionized calcium concentrations fall, CaSR activation decreases—leading to increased PTH release and enhanced renal tubular calcium reabsorption.

The CaSR also binds magnesium, and thus PTH secretion can be inhibited by elevated serum concentrations of magnesium with consequent hypocalcemia. The CaSR may participate in magnesium homeostasis by altering reabsorption of magnesium in the thick ascending limb of Henle in the kidneys. It is probable that increased peritubular levels of magnesium activate renal CaSRs, leading to inhibition of reabsorption of magnesium from the thick ascending limb of Henle—which in turn leads to increased renal excretion of magnesium.

Both loss of function and gain of function mutations in the CaSR have been described in patients with hypocalcemia and hypercalcemia, respectively. Familial (benign) hypocalciuric hypercalcemia type 1(FHH1) and neonatal severe hyperparathyroidism (NSHPT) are caused by loss-of-function mutations of the CaSR gene. Most of these mutations are located in the amino-terminal extracellular domain. With few exceptions, individuals heterozygous for loss-of-function mutations have FHH1, a benign condition characterized by very low urinary calcium excretion, mild hypercalcemia, normal or slightly elevated serum PTH levels, and few if any symptoms of hypercalcemia or hyperparathyroidism. In contrast, individuals homozygous for such mutations will develop NSHPT, a life-threatening condition characterized by severe hypercalcemia, markedly elevated serum PTH levels and skeletal defects. Therefore children of consanguineous FHH parents are at risk for NSHPT. Occasionally, infants with NSHPT are heterozygous for CaSR gene mutations that encode a dominant negative receptor protein. In most cases, FHH1 is transmitted in an autosomal dominant manner, but autosomal recessive inheritance has been described in one kindred in which the CaSR mutation was only weakly inactivating.

Decreased CaSR function impedes calcium ion suppression of PTH release and renal tubular calcium reabsorption. Thus FHH1 is characterized by mild hypercalcemia that is accompanied by inappropriately normal or elevated serum PTH levels and by relatively low urinary calcium excretion. Individuals with FHH1 may also have hypermagnesemia as a result of decreased peritubular inhibition of magnesium reabsorption from the thick ascending loops of the kidneys by the CaSR. In addition to FHH1, there are two other variants, FBH type 2 (FBH2) and FBH type 3 (FBH3), that are caused by heterozygous loss of function mutations in GNA11 and AP2S1, respectively.

FHH1 is caused by heterozygous loss-of-function mutations of the CaSR gene on 3q21.1. Two other chromosomal loci have been identified in patients with FBH who do not have CaSR gene mutations. FHH2 has been mapped to GNA11 , which encodes the α subunit of G11, located 19p13.3 and is biochemically and clinically similar to FHH1. FBH3, which is also known as the Oklahoma variant (FHH _OK ), is caused by mutations in the AP2S1 gene located at 19q13. Adults with FHH3 have hypophosphatemia, elevated serum PTH levels, and osteomalacia, in addition to the clinical and biochemical findings found in individuals with FHH1 and FHH2. NSHPT is characterized by severe hypercalcemia accompanied by elevated circulating PTH levels, undermineralization of bone, rib cage deformity, and multiple long-bone and rib fractures.

Activating mutations of the CaSR gene cause autosomal-dominant hypocalcemia type 1 (ADH1), as increased CaSR function leads to increased calcium ion suppression of PTH release and suppression of renal tubular calcium reabsorption. ADH1 is characterized by hypocalcemia and hypomagnesemia, accompanied by inappropriately normal or increased urinary calcium excretion and inappropriately normal or low serum PTH levels. Patients with ADH1 may be asymptomatic or may present with tetany, muscle cramps, or seizures during infancy or childhood. Similar to inactivating mutations, most activating mutations are located in the amino-terminal extracellular domain. Treatment of patients with ADH1 with activated forms of vitamin D (e.g., calcitriol) is complicated, as normalization of serum calcium levels is associated with worsening of hypercalciuria, hence further increasing the risk of nephrolithiasis, in nephrocalcinosis, and in renal impairment. In contrast, urinary calcium excretion is not excessive in patients with ADH2, who have activating mutations in GNA11 . GNA11 encodes the α subunit of G11, the principle G protein that couples to CaSR in parathyroid cells but not in the kidney.

Some patients with activating mutations of the CaSR gene will develop Bartter syndrome type V, which similar to other types of Bartter syndrome, is characterized by hypokalemic metabolic alkalosis and by hyperaldosteronism caused by elevated renin levels. Patients with Bartter syndrome type V, unlike patients with other types of Bartter syndrome, may also have symptomatic hypocalcemia and are at risk for developing nephrocalcinosis because of hypercalciuria. Evidence from in vitro functional expression studies suggests that patients with mild or moderate heterozygous gain-of-function mutations of the CaSR develop ADH1, whereas those with severe heterozygous gain-of-function mutations of the CaSR will also develop Bartter syndrome type V.

Some single–amino-acid polymorphisms of the CaSR gene appear to predict whole-blood ionized and serum total calcium levels and may increase the risk for bone and mineral metabolism disorders in individuals with other genetic and environmental risk factors for these disorders. Individuals heterozygous or homozygous for a Gln1011Glu CaSR gene polymorphism tend to have higher calcium levels than individuals with the polymorphism. The 15.4% of 387 healthy young Canadian women, with at least one CaSR gene allele with an Ala986Ser polymorphism, were found to have higher total calcium levels than the remainder of the women without the polymorphism.

Another study of 377 unrelated healthy Italian adult males and females found that 24% of study subjects were heterozygous or homozygous for the p.Ala986Ser polymorphism and confirmed the finding that individuals without the polymorphism have lower whole-blood ionized calcium levels than individuals with the polymorphism. The p.Ala986Ser polymorphism has also been associated with Paget disease and primary hyperparathyroidism. Individuals with a less common Arg990Gly polymorphism tend to have lower whole-blood ionized calcium levels than individuals without the polymorphism. The p.Arg990Gly polymorphism has been found to be associated with hypercalciuria and nephrolithiasis.

Autoantibodies against the CaSR that interfere with binding of calcium to the receptor may cause autoimmune hypocalciuric hypercalcemia. These patients have primary hyperparathyroidism with the clinical and biochemical features of patients with FHH1. Conversely, autoantibodies that activate the CaSR are a cause of autoimmune-acquired hypoparathyroidism. Both conditions may occur in association with other autoimmune conditions (such as autoimmune thyroiditis), with celiac disease in patients with autoimmune hypocalciuric hypercalcemia, and with autoimmune thyroiditis and autoimmune polyglandular syndrome types 1 and 2 in patients with autoimmune-acquired hypoparathyroidism. Autoantibodies that activate the CaSR were found in approximately one-third of individuals with acquired hypoparathyroidism.

Inactivating Mutations of the GNAS Gene

Pseudohypoparathyroidism type 1a (PHP1a; Albright hereditary osteodystrophy [AHO]) and pseudopseudohypoparathyroidism (PPHP) are caused by heterozygous inactivating mutations of the GNAS gene that encodes Gα _s . PHP1a is characterized by end organ resistance to PTH with consequent hypocalcemia, hyperphosphatemia, and elevated circulating PTH levels. In addition, patients also manifest resistance to other hormones whose receptors couple to Gs, such as GHRH, TSH, gonadotropin, calcitonin, and hypothalamic neurotransmitters. PHP1a patients also have neurocognitive impairment and obesity that reflect the effect of the Gα _s in the brain. In addition, patients manifest a constellation of developmental defects that have been termed Albright hereditary osteodystrophy and which include heterotopic ossifications, short stature, craniofacial anomalies, and brachydactyly D/E of the hands and feet, characterized by shortened fingers and short fourth and fifth metacarpals. Patients with the associated condition PPHP share the same features of AHO as PHP1a, but do not have hormone resistance. The distinction between these two manifestations of the same gene defect is not stochastic but results from a complex mechanism of genomic imprinting that controls transcription of the GNAS gene. Hence, patients with a GNAS mutation on a maternal allele will develop a more severe form of Gsα deficiency with hormone resistance (i.e., PHP1a), whereas patients with identical mutations on the paternal GNAS allele will have a milder condition with normal hormone responsiveness (i.e., PPHP).

Although most cells express Gsα from both parental alleles, in some cells Gsα expression is suppressed from the paternal GNAS allele. Thus patients with PHP1a develop hormone resistance that is limited to the thyroid, pituitary somatotrophs, and proximal renal tubule cells because in these cells, Gα _s is derived principally from the maternal GNAS allele. Thus patients with maternally inherited inactivating GNAS gene mutations express very little Gα _s in these cells and develop hormone resistance. Because Gsα is not expressed from the paternal allele in imprinted tissues, PPHP patients with paternally inherited inactivating GNAS gene mutations do not experience a deficit in Gsα protein in these cells, as they will have a normal amount of Gsα protein that is produced from the wild-type maternal GNAS allele. Patients with either PHP1a or PPHP express only 50% of the normal amount of Gsα protein in cells in which Gsα transcription is not controlled by the imprinting mechanism, which leads to haploinsufficiency, and likely accounts for the similar features of AHO that occur in these two conditions. Subjects with paternally inherited GNAS mutations have variable features of AHO without hormonal resistance and have been described to have PPHP, progressive osseous heteroplasia (POH), or osteoma cutis, based on the clinical phenotype. The basis for these distinctions is unknown.

Subjects with PHP type 1b (PHP1b; MIM 603233) lack typical features of AHO, but may have mild brachydactyly. PTH resistance is the principal manifestation of hormone resistance, but some patients have slightly elevated serum levels of TSH and normal serum concentrations of thyroid hormones as evidence of partial TSH resistance. It was initially thought that PHP1b is caused by inactivating mutations in the PTHR1 gene. However, no deleterious PTHR1 gene mutations have been found in patients with PHP1b.

Epigenetic defects in imprinting of GNAS are the cause of PHP1b. There are three alternative first exons for GNAS (i.e., NESP55, XLαs, and exon A/B) that splice onto exons 2 through 13 and are associated with differentially methylated regions (DMRs). Most relevantly, the exon A/B DMR is methylated in the maternal germ cells and seems to be the principal control element for transcription from exon 1. Loss of methylation in the DMR upstream of exon A/B of the maternal allele is a consistent finding in patients with PHP1b, and this epigenetic defect accounts for decreased Gsα expression from the affected allele. Most cases of autosomal dominant PHP1b are caused by microdeletions on the maternal allele that include exons 3 to 5 or 4 to 6 of the gene encoding syntaxin-16 ( STX16 ). Three other maternally inherited microdeletions involving NESP55 and AS (delNESP55/ASdel3-4) or AS (delAS3-4) alone have been identified, and these deletions produce a more global disruption of methylation that includes three GNAS DMRs (A/B, XL/AS, and NESP55). The genetic basis for most cases of sporadic PHP1b remains unknown and does not appear to be associated with the GNAS locus. These patients have global epigenetic defects in methylation that affect all three DMRs. In some cases, partial or complete paternal uniparental disomy (UPD) for chromosome 20 has been identified, in which two normal copies of GNAS are both derived from the father. Paternal UPD would predict a near complete deficiency of Gα _s in imprinted cells and tissues in which Gα _s is not transcribed from the paternal allele.

Gsα is not expressed in the renal proximal tubules of these patients because of lack of a GNAS allele with a maternal epigenotype. In contrast, both of the GNAS alleles with paternal epigenotypes are normally expressed in nonimprinted cells in which expression of Gsα from the paternal allele is not suppressed.

Activating Mutations of the GNAS Gene

When a GPCR is activated by a ligand, the activated receptor interacts with the heterotrimeric G protein and allows release of bound GDP from the Gα subunit with replacement by GTP. The GTP-bound Gα dissociates from the Gβγ dimer, and both complexes are free to associate with downstream signal generating molecules. The generation of second messengers is terminated by an intrinsic GTPase within the Gα subunit that acts as a timer; hydrolysis of GTP to GDP inactivates Gα and increases its affinity for Gβγ—leading to reassociation of an inactive heterotrimeric G protein that is ready for another cycle of receptor-induced activation. For Gsα, the amino acids Arg201 and Gln227 are critical to GTPase activity. GNAS mutations that result in substitutions of these amino acid residues lead to abrogation of GTPase activity and therefore prolong the active state of Gα _s , thereby leading to constitutive (i.e., receptor ligand-independent) stimulation of adenylyl cyclase. Somatic mutations of these residues that disrupt GTPase activity are present in approximately 40% of GH–secreting and some ACTH-secreting and nonsecreting pituitary tumors; in some parathyroid, ovarian, testicular, thyroid, and adrenal tumors; and in some intramuscular myxomas.

More widespread mosaic Arg201 Gα _s mutations that decrease GTPase activity cause fibrous dysplasia or (when tissue distribution of the mutation is very widespread) McCune-Albright syndrome, which is characterized by the triad of café-au-lait spots, polyostotic fibrous dysplasia, and primary endocrine hyperfunction, particularly gonadotropin-independent precocious puberty.

Patients with McCune-Albright syndrome may also have excessive GH production, hyperthyroidism, and hypercortisolism, as well as nodularity of the pituitary, thyroid, and adrenal glands because of the growth-promoting effects of excessive cyclic AMP in these tissues. Hypophosphatemia, which is not uncommon in patients with McCune-Albright syndrome, appears to be caused by excessive production of the phosphatonin fiberblast growth factor (FGF)-23 by fibrous dysplasia skeletal lesions. Patients with McCune-Albright syndrome may also have nonendocrine problems, such as hepatobiliary abnormalities, cardiomyopathy, optic neuropathy, and sudden death.

Structure and Function of Type 1 Cytokine Receptors

All type 1 cytokine receptors have four conserved cysteine residues, fibronectin type 2 modules, a Trp-Ser-X-Trp-Ser motif in the extracellular domain, and a proline-rich Box 1/Box 2 region in the cytoplasmic domain. With the exception of stem cell factor, type 1 cytokine receptors do not contain catalytic domains, such as kinases.

Type 1 cytokine receptors for long-chain type 1 cytokines require homodimerization for activation. First, the ligand binds a monomeric receptor. Then, the ligand interacts with a second receptor to induce receptor dimerization and activation. Activated receptors then stimulate members of the Janus family of tyrosine kinases (Jak kinases) to phosphorylate tyrosine residues on both the kinase itself and the cytoplasmic region of the receptors. Signal transducers and activators of transcription (STATs) then dock on the phosphorylated cytoplasmic receptor domains or Jak kinases via an SH2 domain, and are tyrosine phophorylated. The phosphorylated STATs then dissociate from the receptors or Jak kinases, form homodimers or heterodimers, and translocate to the nucleus. In the nucleus, the STAT dimers bind and alter the activity of regulatory regions of target DNA.

There are four Jak kinases. Jak3 is only expressed in lymphohematopoietic cells, whereas Jak1, Jak2, and Tyk2 are expressed in every cell. There are seven STATs (Stat1, Stat2, Stat3, Stat4, Stat5a, Stat5b, and Stat6), which have different SH2 domain sequences that confer different receptor specificities.

Cytokine Receptors That Transduce Hormone Action

The actions of GH, prolactin, and leptin are mediated via specific type 1 cytokine receptors. Mutations of the growth hormone receptor (GHR) and the leptin receptor have been identified as the bases of specific endocrine disorders ( Table 3.3 ).

Fibroblast Growth Factor Receptor 1

Inactivating mutations of the FGFR1 gene are a cause of autosomal-dominant KS. Individuals with KS have anosmia and isolated hypogonadotropic hypogonadism. The FGFR1, which is located on 8p12, plays a role in olfactory and GnRH neuronal migration from the nasal placode to the olfactory bulb, and in the subsequent migration of the GnRH neurons to the hypothalamus. Before identification of these FGFR1 gene mutations, X-linked KS was thought to be caused only by inactivating mutations of the KAL1 gene. KAL1 encodes anosmin-1, the ligand for the FGFR1 receptor. Like FGFR1s, anosmin-1 plays a role in olfactory and GnRH neuronal migration to the nasal placode, and in the subsequent migration of GnRH neurons to the hypothalamus.

There is a high penetrance for anosmia and signs of hypogonadotropic hypogonadism (including lack of puberty, microphallus, and cryptorchidism) in the 10% of KS patients with X-linked KS because of KAL1 gene mutations. Female carriers of KAL1 gene mutations do not have anosmia or isolated hypogonadotropic hypogonadism. In contrast to patients with KS caused by KAL1 gene mutations, the approximately 10% of KS patients with FGFR1 gene mutations (even within the same kindred) exhibit variable phenotypes, ranging from anosmia and complete hypogonadotropic hypogonadism (characterized by cryptorchidism and microphallus in males and absent pubertal development in both genders) to anosmia or delayed puberty.

It has also been noted that in most kindreds with FGFR1 gene mutations, females present with more mild KS phenotypes than males. Female carriers may even be asymptomatic. Because the KAL1 gene is located on the X chromosome, females may produce more anosmin-1 than males. Thus a possible explanation for milder KS phenotypes in females with FGFR1 gene mutations may be that the increased anosmin-1 levels in females may lead to increased anosmin-1–induced activation of the mutant FGFR1s that may partially compensate for the mutation. Interestingly, missing teeth and cleft palate are not an uncommon finding in individuals with KS resulting from FGFR1 gene mutations, whereas unilateral renal agenesis and bilateral synkinesia are associated with KS arising from KAL1 gene mutations.

Fibroblast Growth Factor Receptors 2–4

A diverse group of skeletal disorders are caused by activating mutations in the FGFR1 gene, as well as in the related genes encoding FGFR2 and FGFR3. In general, gain-of-function mutations in FGFR1 and FGFR2 cause most of the syndromes involving craniosynostosis, whereas the dwarfing syndromes are largely associated with FGFR3 mutations. Osteoglophonic dysplasia is a “crossover” disorder that has skeletal phenotypes usually associated with FGFR1, FGFR2, and FGFR3 mutations. Osteoglophonic dysplasia is caused by missense mutations in highly conserved residues comprising the ligand-binding and transmembrane membranes of FGFR1, thus defining novel roles for this receptor as a negative regulator of long bone growth. Gain-of-function mutations in genes encoding the other FGF receptors cause related skeletal disorders: Pfeiffer syndrome (activating mutations of FGFR1 and FGFR2), Crouzon syndrome (gain-of-function FGFR2 mutations), Crouzon syndrome with acanthosis nigricans (an FGFR3 mutation), Apert syndrome (FGFR2 mutations), and craniosynostosis (gain-of-function FGFR3 mutations). Several autosomal-dominant short-limb dwarfism syndromes—including achondroplasia, severe achondroplasia with developmental delay and acanthosis nigricans (SADDAN), hypochondroplasia, and three types of platyspondylic lethal skeletal dysplasias (PLSD) (thanatophoric dysplasia I [TDI], thanatophoric dysplasia II [TDII], and San Diego types [PLSD-SD])—are often caused by heterozygous constitutively activating FGFR3 gene mutations.

Individuals with achondroplasia have activating mutations in the transmembrane domain of FGFR3, with the p.Gly380Asn found in more than 95% of achondroplastic patients. From 40% to 70% of individuals with hypochondroplasia have an activating p.Asn540Lys mutation in the TK1 domain. All individuals with TDII have an activating p.Lys650Glu mutation in the activating loop of the TK2 domain, and more than 90% of individuals with TDI and PLSD-SD have FGFR3 mutations. Patients with SADDAN have an activating mutation in the same codon as patients with TDII. Instead of the p.Lys650Glu mutation associated with TDII, patients with SADDAN have a Lys650Met mutation. However, unlike patients with TDII patients with SADDAN do not have craniosynostosis and a cloverleaf skull—and often survive past childhood.

The FGFR3 gene is primarily expressed in endochondral growth plates of long bones, brain, and skin pre- and postnatally. Constitutional activation of FGFR3s in chondrocytes leads to growth arrest and apoptosis. In addition, constitutive activation of FGFR3s is also postulated to alter neuronal migration because patients with SADDAN, TDI, and TDII have neurologic abnormalities that may include developmental delay, paucity of white matter, polymicrogyria, dysplastic temporal cortex, dysplasia of nuclei, and neuronal heterotopia. Furthermore, constitutive activation of FGFRs in skin fibroblasts and keratinocytes is thought to cause the acanthosis nigricans seen in patients with SADDAN and Crouzon syndrome with acanthosis nigricans. However, it is not yet known why some activating FGFR3 mutations affect the skeletal system, central nervous system, and the skin, whereas other activating FGFR3 mutations only affect the skeletal system.

Loss-of-function mutations in FGFR3 also have been associated with human disease. An uncommon syndrome characterized by camptodactyly, tall stature, scoliosis, and hearing loss (CATSHL syndrome) has been associated with a heterozygous missense mutation (p.R621H) in the TK domain and partial loss of FGFR3 function. These findings indicate that abnormal FGFR3 signaling can cause human anomalies by promoting, as well as inhibiting, endochondral bone growth.

General Structure of the Nuclear Receptors

Nuclear receptors are made up of four domains: A/B, C, D, and E ( Fig. 3.9 ). Supporting the notion that nuclear receptor subfamilies are derived from a common ancestral orphan receptor, the C and E domains are highly conserved among the subfamilies. Mutations of several nuclear receptors are associated with endocrine disorders ( Table 3.5 ).

Ligand-induced activation of transcription by nuclear receptors. Often, corepressors, including silencing mediator of retinoid and thyroid hormone receptors (SMRT) and nuclear receptor corepressor (N-CoR) , bind a nuclear receptor that is not bound by its ligand. These corepressors then associate with Sin3, which in turn associates with a histone deacetylase (HDAC) . Then, HDAC represses transcription by deacetylating histone tails—resulting in compaction of the nucleosomes into structures that are inaccessible to transcription factors. Ligand binding induces structural changes in the E domain that result in release of the corepressor/Sin3/HDAC complexes from the receptor and binding of coactivator complexes that may include steroid receptor coactivator 1 (SRC-1) , p300/cyclic adenosine monophosphate responsive element binding protein (CBP) , p300/CBP-associated factor (P/CAF), or p300/CBP cointegrator-associated protein (pCIP) to the LXXLL motif of the AF2-AD. Then the coactivator complexes induce transcription by acetylating (Ac) the histone tails—resulting in decompaction of the nucleosomes into structures that are accessible to transcription factors. Dashed lines are used to represent coactivator and corepressor complexes because theircomposition in vivo is yet unknown.

(Modified from Robyr, D., Wolffe, A. P., & Wahli, W. (2000). Nuclear hormone receptor coregulators in action: diversity for shared tasks. Mol Endocrinol , 14, 339. Copyright 2000, The Endocrine Society. With permission.)

The A/B domain is located at the amino-terminal and contains the activation function 1 (AF-1)/τ 1 domain. The AF-1/τ 1 domain regulates gene transcription by interacting with proteins (such as the Ada and TFIID complexes) that induce transcription. This transactivation function of the AF-1/τ 1 domain is not dependent on binding of the nuclear hormone receptor to its ligand and is not specific in its choice of DNA target sequences. Thus specificity of action of the nuclear hormone receptor is determined by the function of other nuclear hormone receptor domains.

The C domain has characteristics that help to confer specificity of action on each nuclear hormone receptor. This domain consists of two zinc-finger motifs responsible for the DNA-binding activity of the receptor and the selection of dimerization partners. Each zinc-finger module consists of a zinc ion surrounded by the sulfurs of four cysteine residues, resulting in a tertiary structure containing helices. The P-box lies near the cysteines of the first zinc finger and contains the three to four amino acids responsible for specificity of binding to response elements. The D-box consists of a loop of five amino acids attached to the first two cysteines of the second zinc finger that provides the interface for nuclear receptor dimerization.

The D “hinge” domain contains nuclear localization signals and contributes to the function of the adjacent C and E domains. Thus the amino-terminal portion of the domain contributes to DNA binding and heterodimerization and the carboxy-terminal portion contributes to ligand binding. The nuclear localization signal plays a particularly important role in the function of glucocorticoids and mineralocorticoid receptors because these receptors bind their ligand in the cytoplasm and must then localize to the nucleus to alter gene transcription.

The E domain is known as the ligand-binding domain ( LBD ) or the hormone-binding domain . In addition to ligand binding, the E domain has effects on dimerization and transactivation. The LBD consists of 11 to 12 α helices (named H1 through H12) and contains a ligand-binding pocket that is made up of portions of some of the different helices. For example, the thyroid receptor (TR) LBD has a ligand-binding cavity that includes components from H2, H7, H8, H11, and H12. The contribution of different parts of LBD to the ligand-binding pocket accounts for the finding that mutation of single–amino-acid molecules in different helices of the LBD can interfere with ligand binding.

Unlike the AF-1/τ 1 transcriptional activating factor, the E domain activation factor 2 (AF2-AD) requires ligand binding to function (see Fig. 3.9 ). Often, when the receptor is not bound by its ligand, corepressor complexes simultaneously bind the LBD and transcriptional machinery consisting of protein complexes that place transcription factors on nucleosome binding sites (see Fig. 3.9 ). The corepressor complexes then suppress gene transcription by using histone deacetylases to compact the nucleosomes into inaccessible structures (see Fig. 3.9 ). Ligand binding induces structural rearrangements in the E domain that lead to release of these corepressor complexes from the transcriptional machinery and the LBD, and exposure of the transcriptional machinery and the LXXLL motif of the AF2-AD to coactivator complexes (see Fig. 3.9 ). These coactivator complexes have histone acetyltransferase activity that acts to relax nucleosome structures, enabling transcription factors to access nucleosome binding sites (see Fig. 3.9 ).

Most nuclear receptors are capable of binding their hormone response element and repress transcription when they are not bound by their ligand. However, in the absence of ligand, steroid receptors are bound to a complex of heat-shock proteins instead of their response element and do not appear to repress transcription.

Agonists and antagonists have different effects on the interaction between the ligand binding pocket and AF2-AD. For example, when 17β-estradiol binds to the estrogen receptor, the position of the AF2-AD containing H12 is altered so that coactivators can access the LBD-binding coactivator binding site. However, when the estrogen antagonist raloxifene binds at the same site, the coactivator binding site on H12 remains blocked by other portions of H12.

Although some nuclear receptors are fully active when bound as monomers to DNA, the hormone receptors in the nuclear receptor superfamily are most active when bound as homodimers or heterodimers (see Fig. 3.9 ). RXRs, HNF 4, and the steroid hormone receptors can bind DNA as homodimers or heterodimers. The α isoform of the estrogen receptor (ESR1) is particularly promiscuous, and is able to heterodimerize with HNF4A and retinoic acid receptors, the β isoform of the estrogen receptor (ESR2), RXR, and the thyroid hormone receptors. As a homodimer, RXR binds the dINSRect repeat 1 (DR1). It can also join the thyroid, vitamin D ₃ , and peroxisome proliferator–activated receptors to form heterodimers.

Interestingly, it has also been suggested that some steroid hormones also act on transmembrane receptors—and these interactions may be responsible for the acute cellular effects of steroids. Progesterone has been shown to interact with the G protein–coupled uterine oxytocin, nicotinic acetylcholine, γ-aminobutyric acid _A , N -methyl- D aspartate, and sperm cell membrane progesterone receptors. Cell membrane estrogen and glucocorticoids receptors have also been identified.

Thyroid Hormone Receptors

The two thyroid hormone receptor (THR) isoforms—thyroid hormone receptor α (THRA) and thyroid hormone receptor β (THRB)—are encoded by different c-erbA genes on chromosomes 17 and 3, respectively. Alternate splicing leads to expression of TRα1, TRα2, TRβ1, and TRβ2 with different tissue distributions. TRα2 does not bind thyroid hormone and its function is not understood. TRα1 is the predominant subtype in cardiac and skeletal muscle, bone, gastrointestinal tract, and the central nervous system. TRβ1 is the predominant subtype in kidney and liver, whereas TRβ2 is expressed in hypothalamus and pituitary, as well as retina and cochlea. THRs that are not occupied by the thyroid hormone triiodothyronine (T ₃ ) exist as homodimers or heterodimers, with RXRs that are attached to DNA thyroid hormone response elements in association with corepressor proteins. Thyroid hormone binding induces the release of the corepressors from the THR. A coactivator, steroid receptor coactivator-1 (SRC-1), is then able to attach to the THR—enabling activation of transcription.

Generalized resistance to thyroid hormones (GRTH) can be autosomal recessive or autosomal dominant, as the presence of a single normal THRB allele is sufficient for normal receptor function. Autosomal dominant GRTH is caused by the presence of an abnormal THRB that interferes with the function of the normal receptor in a dominant-negative fashion. The prevalence of dominant GRTH, based on newborn screening, was reported to be 1:40,000 live births. Patients with this syndrome have an impaired receptor response to T ₃ . They have elevated T ₃ , reverse T ₃ , and thyroxine (T ₄ ) levels with slightly high TSH levels. Consistent with the TSH elevation and goiter, serum thyroglobulin levels tend to be elevated and radioiodine uptake high. Unlike autoimmune thyrotoxicosis, the T ₃ : T ₄ ratio is normal. The clinical manifestations are variable including some hyperthyroid and some hypothyroid symptoms. Findings include goiter in 65% to 85% of those affected, hyperactivity in 33% to 68%, tachycardia in 33% to 75%, failure to thrive, raised metabolic rate, low bone density, hearing defects, learning disabilities, developmental delay, and delayed bone age. GRTH can be detected by newborn screening if the program measures T4, in addition to or in lieu of TSH only, as T ₄ would be elevated and TSH would be elevated. The combination of findings normally associated with hyperthyroidism and hypothyroidism is caused by differential expression of TRα versus TRβ. The hypothalamus and pituitary express TRβ and this leads to elevated TSH levels. Cardiac tissues express TRα and this leads to tachycardia as elevated T ₄ and T ₃ act on the normal TRα receptor.

Mutations have been found in the D and E domains of THRBs of GRTH patients. These mutations alter ligand binding or transactivation. However, most mutant THRBs retain the ability to repress transactivation of target genes through interactions with corepressors. Some of the GRTH mutant receptors continue to associate with corepressors and are unable to bind the coactivator SRC-1 even when bound by T ₃ . Thus mutant THRBs have a dominant negative effect in the heterozygous state because they are able to interfere with the function of wild-type receptors by repressing transcription of target DNA.

Patients who carry biallelic loss-of-function mutations of THRB demonstrate more severe clinical abnormalities than patients with heterozygous dominant negative mutations. One patient with a deletion of both THRB alleles presented with deaf mutism, dysmorphic features, and stippled epiphyses. Another patient homozygous for a THRB mutant (“kindred S receptor”), with an amino acid deletion in the ligand-binding domain, presented with mental retardation, very delayed bone age, and very elevated T ₃ and T ₄ levels. Heterozygous carriers of the kindred S receptor mutation have milder clinical manifestations of GRTH because this mutant THRB retains corepressor activity and thus has dominant negative effects.

Mutations in THRA were not identified until recently. The first described proband is heterozygous for a nonsense mutation (p.E403X) that inhibits the wild-type receptor in a dominant negative fashion. The patient had hypothyroid features (growth retardation, developmental delay, skeletal dysplasia, and constipation) that reflect the distribution of target tissues in which THRA is expressed. This is associated with borderline low or normal T ₄ and free T ₄ , low reverse T ₃ , borderline high or normal T ₃ and free T ₃ , a low T ₄ : T ₃ ratio, and normal TSH. After thyroxine treatment, normalizing T ₄ levels, T ₃ levels became elevated. It has been suggested that this may be related to increased type 1 deiodinase activity (which converts T ₄ to T ₃ ) and reduced type 3 deiodinase activity (which converts T ₄ to reverse T ₃ and T ₃ to T ₂ ). The mutant receptor failed to bind radiolabeled T ₃ and failed to activate a thyroid hormone responsive reporter gene and inhibited the activity of the wild-type receptor. The second report described a one base insertion in a father and a daughter, causing a frameshift and premature termination at codon 406. Analyses of the mutant receptor in vitro, after expression in cultured cells, revealed that the mutant receptor fails to respond to stimulation by T ₃ and exerts a strong dominant negative effect on the wild-type receptor. This and other subsequent reports identified more clinical features, such as macrocephaly, dysmorphic facies (round, flat face with course features) increased birth length and weight, anemia, and slightly elevated cholesterol. Several reports have recently documented different mutations, including missense mutations with partially active receptors. These studies demonstrated that the severity of clinical findings correlated with the degree of receptor impairment. The p.A263V mutation exhibited impaired transcription at low T ₃ concentrations, but was active at higher T ₃ concentrations and was associated with a milder phenotype. Another mutation (p.N359Y), which affects both TRα1and TRα2, was associated with additional clinical features, namely, micrognathia, clavicular agenesis, metacarpal fusion, syndactyly, hyperparathyroidism, and chronic diarrhea.

Vitamin D Receptor

Severe rickets, hypocalcemia, secondary hyperparathyroidism, and increased 1,25-dihydroxyvitamin D (calcitriol) levels occur in patients with the autosomal recessive syndrome of “vitamin D resistance.” These patients have defective vitamin D receptors (VDRs). Mutations causing this syndrome have been found in the zinc fingers of the DNA-binding domain (C domain), leading to decreased or abolished receptor binding to regulatory elements of target genes. Causative mutations have also been found that lead to the production of receptors that have decreased or abolished ability to bind calcitriol and heterodimerize with RXRs, which are required for the VDR to maximally transactivate target genes.

Less severe mutations in the VDR are associated with decreased gastrointestinal calcium absorption and bone mineral density, even during childhood, and an increased risk for osteoporosis and fractures. However, it has not been possible to replicate these findings in some ethnic groups. Thus other factors (such as estrogen receptor genotype, dietary calcium, and age) probably contribute to the effects of VDR polymorphisms on bone mineral metabolism. Certain VDR polymorphisms are associated with decreased pre- and postnatal linear growth.

Other important associations have been found with VDR polymorphisms. Homozygous polymorphisms have been reported to be associated with primary hyperparathyroidism. In addition, the presence of certain VDR alleles is associated with increased risk for the development of early-onset periodontal disease. Conversely, absence of such alleles has been associated with familial calcium nephrolithiasis. Absence of certain alleles may be a risk factor for the development of sarcoidosis. However, the presence of these alleles is associated with hypercalciuria and nephrolithiasis and increased risk in women for the development of metastatic breast cancer. VDR polymorphisms have also been associated with increased susceptibility to psoriasis, tuberculosis, leprosy, and other infections.

Peroxisome Proliferator-Activating Receptor γ

Peroxisome proliferator-activating receptor γ (PPARγ) has a role in regulating adipocyte differentiation and metabolism. This formerly orphan receptor was adopted by the ligand prostaglandin J2. Mutations in the gene encoding PPARγ cause familial partial lipodystrophy type 3 ( FPLD3 ).

Patients with FPLD3 exhibit fat loss in the arms and legs, severe insulin resistance, early-onset type 2 diabetes mellitus, and severe hypertriglyceridemia. Some heterozygous mutations have a dominant negative effect on the wild-type receptor (V290M and P467L). These mutations lead to amino acid substitutions that disturb the orientation of H12 in the E domain, leading to decreased ligand-dependent transactivation by AF-2/AD and coactivator recruitment. Other mutations are point mutations that cause FPLD3 simply because of haploinsufficiency without a dominant negative effect. In addition, a frameshift mutation that causes a similar phenotype was identified. Another mutation in PPARγ expanded the phenotype to include muscular, immune, and hematologic features. This mutation is thought to induce a conformational change affecting transcriptional activation by the receptor.

A missense mutation leading to a p.Pro115Gln substitution near a site of serine phosphorylation at position 114 that suppresses transcriptional activation in PPARγ2 was found in some morbidly obese patients. The p.Pro115Gln substitution interferes with phosphorylation of the serine at position 114, leading to increased transcriptional activation by PPARγ2—which in turn leads to increased adipocyte differentiation and triglyceride accumulation.

Hepatocyte Nuclear Factor Receptors

Alteration of another orphan nuclear receptor, HNF4A, also causes an endocrine disorder. Mutations of the HNF4A gene on chromosome 20 that alter the ligand-binding domain (E domain) or the DNA-binding domain (C domain) have been found in patients with a form of monogeneic diabetes, also known as maturity-onset diabetes of the young type 1 (MODY1). Patients with MODY usually develop diabetes mellitus by the end of the third decade of life. They have a defect in glucose-mediated stimulation of insulin secretion. Several studies have now described a biphasic phenotype of neonatal hyperinsulinism and macrosomia, with later onset of diabetes in patients with HNF4A mutations. In most cases, the hyperinsulinism is transient but can be persistent, requiring diazoxide treatment.

Patients who are heterozygous for the p.R76W HNF4A mutation also develop a Fanconi-Bickel like syndrome in addition to neonatal hyperinsulinism and macrosomia. The patients manifest the typical proximal tubulopathy of Fanconi-Bickel syndrome, including generalized aminoaciduria, low-molecular-weight proteinuria, glycosuria, hyperphosphaturia, and hypouricemia, plus additional features, including nephrocalcinosis, renal impairment, hypercalciuria with relative hypocalcemia, and hypermagnesaemia. The Fanconi-Bickel like phenotype has been recapitulated in a mouse model in which genetic ablation of Hnf4a was targeted to the kidney, indicating that Hnf4a is required for the formation of differentiated proximal tubules and that loss of Hnf4a decreased the expression of proximal tubule-specific genes. The p.R76W mutation occurs in the DNA-binding domain of HNF4A, and has been hypothesized to cause defective interaction with major regulatory genes; it remains unknown why patients with other HNF4A mutations do not develop the same renal phenotype.

Carriers of a glycine-to-serine substitution in codon 115 in the DNA-binding domain (C domain) appear to be at increased risk for developing low-insulin diabetes mellitus. Hepatocyte nuclear factors 3 (HNF-3α, -3β, and -3γ) are also regulators of the early-onset type 2 diabetes genes HNF1A , HNF4A , and IPF-1/PDX-1 —which are associated with MODY types 3, 1, and 4, respectively.

Glucocorticoid Receptors

Glucocorticoids strongly influence cardiovascular tone; have actions on liver, muscle, and adipose tissue; and exert potent antiinflammatory and immunosuppressive effects. Glucocorticoids are important in growth and development, as well as behavior and cognition. They influence multiple cellular processes, including proliferation, differentiation, and apoptosis. All of these actions are mediated by the glucocorticoid receptor (GR), which is a ligand activated transcription factor influencing transcription of approximately 20% of the genome. The gene for the glucocorticoid receptor is known as NR3C1 . NR3C1 is on chromosome 5 and consists of 10 exons. Multiple isoforms of the GR exist because of alternative splicing, insertions, deletions, and alternative translation initiation. The two main forms are hGRα and hGRβ. hGRβ does not bind natural or synthetic glucocorticoids; is expressed in few cell types, such as neutrophils and epithelial cells; and may exert inhibitory effects on the main glucocorticoid receptor hGRα. hGRα is ubiquitously expressed in all tissue but the suprachiasmatic nuclei of the hypothalamus.

Mutations in glucocorticoid receptors cause primary generalized glucocorticoid resistance (PGGR) or primary generalized glucocorticoid hypersensitivity (PGGH). PGGR is clinically characterized by the presence of elevated plasma cortisol and ACTH levels, accompanied by the effects of hyperaldosteronism and hyperandrogenism in the absence of striae or central fat deposition. ACTH excess leads to adrenocortical hyperplasia, increased cortisol secretion, and increased production of adrenal steroids with androgenic (androstenedione, dehydroepiandrosterone [DHEA], DHEA-S) and mineralocorticoid activity (cortisol, deoxycorticosterone and corticosterone). The clinical spectrum is broad, ranging from asymptomatic to severe hyperandrogenism (characterized by severe acne, hirsutism, irregular menses, and infertility), mineralocorticoid excess (characterized by hypokalemic alkalosis and hypertension), and fatigue. Girls may present with ambiguous genitalia and both girls and boys may present with isosexual precocity. Clinical manifestations of glucocorticoid deficiency are infrequent and largely limited to fatigue. However, there were reports of childhood hypoglycemia.

Both homozygous and heterozygous mutations have been described. Heterozygous mutations that cause PGGR generally do so by exerting a dominant negative effect on the wild-type receptor.

ACTH-secreting pituitary macroadenomas can also be caused by a frameshift mutation in the GR gene that interferes with signal transduction. Patients with this mutation manifest the symptoms of glucocorticoid resistance. The tumor develops as a result of impaired negative feedback regulation by glucocorticoids on the hypothalamic-pituitary axis.

PGGH was described in a 43-year-old female who presented with visceral obesity, hypertension, hyperlipidemia, and type 2 diabetes, and was found to have a mutation in the GR (D401H) that demonstrated increased transactivation of glucocorticoid responsive genes. The patient had evidence of glucocorticoid resistance in the hypothalamus–pituitary gland–adrenal gland axis and hypersensitivity at the vasculature, adipose tissue, and liver. This was associated with an elevated ACTH and am cortisol but normal urine free cortisol.

Androgen Receptors

The human androgen receptor ( AR ) gene is located on the X chromosome. Known disorders characterized by AR dysfunction caused by AR gene mutations are only expressed in patients with a 46 XY karyotype. These mutations may be transmitted from an asymptomatic carrier mother or can be de novo.

More than 1000 mutations have been described in the AR gene, with more than 500 mutations causing androgen insensitivity syndrome (AIS). The phenotype of this syndrome can vary in severity and has been divided into mild, partial, and complete forms (MAIS, PAIS, and CAIS). CAIS is characterized by intraabdominal testes, absence of mullerian structures, absence of androgen-induced body hair, such as pubic and axillary hair, and a female appearance. PAIS refers to individuals with ambiguous external genitalia with an enlarged clitoris or microphallus and patients with Reifenstein syndrome. Reifenstein syndrome is characterized by severe hypospadias with scrotal development and severe gynecomastia. MAIS refers to patients with AR mutations who are otherwise normal phenotypic males who present with adolescent gynecomastia or later infertility.

The phenotypic heterogeneity of AIS is caused by the varied locations of the mutations causing AIS. Functional consequences of each mutation causing AIS relate to the function of the domain in which the mutation is located. However, the degree of impairment of mutated receptor function in in-vitro studies does not always correlate with the phenotypic severity of the syndrome.

Mutations in exons that code for the AR hormone-binding domain decrease hormone-binding affinity. However, these mutations do not abolish the hormone-binding capability of the receptor. Thus patients with these mutations usually present with PAIS or occasionally with CAIS. Patients with mutations in the hormone-binding domain do not appear to respond to treatment with high doses of testosterone. Mutations in the DNA-binding domain lead to failure of target gene regulation. Thus patients with these mutations usually manifest the CAIS.

CAIS is also caused by a point mutation that results in a premature termination codon (p.Gln340ter), with apparent initiation of translation that is downstream of the termination codon. In vitro studies showed that the truncated AR is expressed at reduced levels. Numerous other mutations have been described that cause truncation or deletion of the AR and complete AIS. Two patients with ambiguous genitalia and partial virilization were found to be mosaic for mutant ARs.

Some patients with Reifenstein syndrome have been found to have a mutation in the DNA-binding domain that abolishes receptor dimerization. Other patients have been found to have a mutation in a different area of the DNA-binding domain that does not affect receptor dimerization, or to have mutations in the hormone-binding domain in the E domain. The Ala596Thr mutation in the D-box area of the DNA-binding domain has been associated with an increased risk of breast cancer.

AIS is also a feature of Kennedy disease, which is an X-linked recessive condition causing spinal and muscular atrophy. This condition is caused by extension of a poly-CAG segment in the AR gene exon that codes for the N-terminus of the AR, leading to an increased number of glutamine residues in the A/B domain. ARs with a polyQ region increased to 48 glutamine residues accumulate abnormally in transfected cells because of misfolding and aberrant proteolytic processing. Because polyQ extension does not completely abolish transactivation, patients with Kennedy disease exhibit a mild partial AIS phenotype consisting of normal virilization accompanied by testicular atrophy, gynecomastia, and infertility.

Gain-of-function mutations in the AR have been described in prostate, breast, testicular, liver, and laryngeal cancers.

Estrogen Receptors

Two major full-length ESR isoforms have been identified in mammals. Estrogen receptor α (ESR1) was discovered first and mediates most of the known actions of estrogens. ESR1 is expressed primarily in the uterus, ovaries, testes, epididymis, adrenal cortices, and kidneys. Estrogen receptor β (ESR2) was discovered in 1996. ESR1 and ESR2 share 95% and 50% homology in the DNA-binding domain (DBD) and LBD, respectively. There is little homology in the amino-terminal between the two isoforms. ESR2 is expressed primarily in the uterus, ovaries, testes, prostate, bladder, lung, and brain. Although ESR2 has a high affinity for estrogens, it has less transactivating ability than ESR1 and has not yet been found to be involved in any pathologic condition.

There is evidence supporting the existence of other functional ESRs. Some of these putative ESRs localize to the cell membrane instead of, or in addition to, the nucleus. A 46-kDa amino-terminal truncated product of ESR1, named ER46, localizes to the cell membrane and mediates estrogen actions that are initiated at the cell membrane. Another of these putative ESRs has been named ER-X and is postulated to be a GPCR that localizes to the cell membrane. Another putative receptor is the aptly named heterodimeric putative estrogen receptor (pER), which has been found on the cell and nuclear membranes. The pER acts as a serine phosphatase. Five other estrogen-binding proteins have also been identified, and at least three of them localize to the cell membrane.

It had been thought that androgens provide the principal signals for closure of the epiphyses during puberty. In 1994, however, extensive studies of a 28-year-old man, with incomplete closure of epiphyses and tall stature, demonstrated that he was homozygous for a premature termination mutation of codon 157 in exon 2 of the ESR1 gene. The patient had continued linear growth despite otherwise normal pubertal development, demonstrating that the ESR mediates epiphyseal closure. Expression of this gene leads to the production of a nonfunctional ESR1 lacking both the DNA- and hormone-binding domains. He was also found to have increased estradiol levels, impaired glucose tolerance with hyperinsulinemia, and decreased bone density.

Further strengthening the association between ESR1 and epiphyseal closure is the observation that women with ESR1-positive breast cancer and a mutation in the B domain (B’ allele) of ESR1 have an increased incidence of spontaneous abortion and tall stature. These associations were not found in female carriers of the allele without breast cancer or with ESR1-negative breast cancer. Thus a second (as yet undiscovered) mutation is likely to play a role in the development of tall stature and spontaneous abortions in female carriers with ESR1-positive breast cancer.

Mineralocorticoid Receptors

Mineralocorticoid resistance is also known as pseudohypoaldosteronism (PHA). Both sporadic and familial cases with either autosomal-dominant or autosomal-recessive cases have been reported. Clinical presentation of patients with PHA ranges from asymptomatic salt wasting; to growth failure; to chronic failure to thrive, lethargy, and emesis; to life-threatening dehydration accompanied by severe salt wasting. Patients with the severe forms of PHA typically present within a year of birth and may even present in utero with polyhydramnios because of polyuria. Biochemically, the condition is characterized by urinary salt wasting, hyponatremia, elevated plasma potassium, aldosterone, and renin activity, and urinary aldosterone metabolism that are unresponsive to the mineralocorticoid treatment.

Two forms of PHA are recognized (types I and II). Autosomal recessive (generalized) PHAI results from mutations in the epithelial sodium channel (ENaC), whereas autosomal dominant (renal) PHAI is caused by mutations in the mineralocorticoid receptor (MR). PHAII is the result of mutations in a family of serine-threonine kinases known as WNK1 and WNK4 , which are downstream of aldosterone action. PHAI is characterized by renal tubular mineralocorticoid resistance, whereas PHAII results from mineralocorticoid resistance in the kidney, intestine, or salivary or sweat gland and is also known as Gordon syndrome . Patients with these conditions present with hyperkalemia that only responds to treatment with nonchloride ions, such as bicarbonate or sulfate, which increase delivery of sodium to the distal tubule. In general, patients with autosomal dominant PHA1 have a mild salt-wasting syndrome and improve with age, whereas patients with autosomal recessive PHA1 have severe salt wasting and hyperkalemia, as well as increased sweat and salivary sodium and frequent respiratory tract infections that fails to improve with age.

More than 50 different mutations of the MR causing autosomal dominant PHAI have been described (all heterozygous). There is clear heterogeneity of clinical manifestations within families. Haploinsufficiency caused by mRNA or protein degradation is clearly sufficient to cause autosomal dominant PHAI. Yet some mutations have been shown to exert dominant negative effects on the wild-type receptor.

Genetic disorders that are associated with increased activity of the MR cause severe early-onset hypertension. One such condition, characterized by excessive MR action, is termed the syndrome of apparent mineralocorticoid excess . Patients with this autosomal-recessive condition can exhibit pre- and postnatal growth failure, hypervolemic hypertension, medullary nephrocalcinosis, and hypokalemic metabolic alkalosis accompanied by hyporeninemic hypoaldosteronism. Patients may also be asymptomatic and exhibit only biochemical abnormalities. Patients with this syndrome also have increased serum and urinary cortisol-to-cortisone ratios. This syndrome is caused by mutations in the 11 β-hydroxysteroid dehydrogenase type 2 ( HSD11B2 ) gene that reduce enzymatic activity of the protein. The 11 β-HSD2 converts the active glucocorticoid cortisol to inactive cortisone; in contrast the type I isoform, encoded by the separate HSD11B1 gene, has both 11-β dehydrogenase activity and 11-oxoreductase activity and can also convert cortisone to cortisol. The type I isoform is ubiquitously expressed, whereas the type II isoform has more restricted expression and is highly expressed in the kidney, where it is necessary to protect the renal MR from normally higher serum concentrations of cortisol, allowing aldosterone to regulate sodium homeostasis. Thus decreased activity of 11 β-HSD2 increases cortisol levels in kidney cells expressing MR tissues, leading to increased binding and activation of MRs by cortisol.

A transient form of mineralocorticoid resistance, probably because of abnormal maturation of aldosterone receptor function, also exists. This variant of PHA is known as the syndrome of early-childhood hyperkalemia . Children with this disorder present with failure to thrive or linear growth failure accompanied by hyperkalemia and metabolic acidosis. This condition resolves spontaneously by the second half of the first decade of life.

DAX1

The dosage-sensitive sex-reversal adrenal hypoplasia congenital critical region on the X chromosome gene 1 ( DAX1 ) is an orphan nuclear receptor because its ligand has not yet been identified. It has homologies in the E domain to other orphan receptors, including RXRs. However, DAX1 has an unusual DNA-binding C domain that contains a tract of amino acid repeats instead of zinc-finger motifs. DAX1 inhibits SF-1-mediated transcription. SF-1 is another orphan nuclear receptor that regulates transcription of adrenal and gonadal steroid hydroxylases, mullerian-inhibiting substance, and gonadotropin genes.

DAX1 gene mutations have been identified that cause X-linked adrenal hypoplasia congenita. Patients with this condition have congenital adrenal insufficiency and are therefore deficient in glucocorticoid, mineralocorticoid, and androgen production. Some 40% of affected male patients present in the first 2 months of life with a salt-wasting adrenal crisis, and the remainder present later in childhood with mineralocorticoid insufficiency, resembling aldosterone synthase deficiency or PHA. Those who present with adrenal crisis can have hyperkalemia, hyponatremia, hypotension, or cardiac arrest, because of both mineralocorticoid and glucocorticoid deficiency in the setting of normal 17-hydroxyprogesterone levels. Patients may have normal cortisol levels. Imaging of the adrenal by any modality at this age is not helpful, as the condition affects the definitive zone but not the fetal zone of the adrenal. The fetal zone is quite large at birth and does not atrophy until 6 to 12 months of age. Imaging cannot differentiate the fetal from the definitive zones and reports a normal adrenal gland in the first few months of life. Imaging after the first year of life would be expected to show a hypoplastic adrenal. Gonadotropin deficiency and azoospermia also occur in these patients but this does not present until puberty. Patients have findings consistent with hypogonadotropic hypogonadism (because of a combined hypothalamic/pituitary defect) and may have absent pubertal development or puberty that arrests at around Prader stage 3. Female carriers may have delayed puberty. All mutations that have been found to cause X-linked congenital adrenal hypoplasia are either located in or prevent transcription of the area of the E domain that inhibits SF-1-mediated transcription. Thus DAX1 mutations may cause X-linked congenital adrenal hypoplasia by altering SF-1 regulation of gonado- and adrenogenesis. DAX1 deletion may also occur in the setting of the contiguous gene deletion syndrome, resulting in complex glycerol kinase deficiency (cGKD) if individuals have deletions extending from the GK gene into the Duchene muscular dystrophy ( DMD ) gene, or involving a significant extension telomeric from DAX1.

Duplications of Xp that include DAX1 in genetic males with XY karyotype lead to sex reversal. DAX1 overexpression represses all aspects of male differentiation beginning with the urogenital ridge progressing to a bipotential gonad, under the influence of WT1 and SF1, to differentiation into a testis under the influence of SF1, SOX9, SRY, and AMH and leads to female external genitalia with variable female internal organs from absent ovaries, uterus, and cervix to normal ovaries with rudimentary mullerian structures. Duplications in this region of the X chromosome are also associated with developmental delay, low-set ears, cleft palate, clinodactyly, hypotonia, thin upper lip, simian creases, and cardiac abnormalities.