Chapter 66 – Pseudohypoparathyroidism, Albright’s ...66 PSEUDOHYPOPARATHYROIDISM, ALBRIGHT’S HEREDITARY OSTEODYSTROPHY, AND PROGRESSIE OSSEOUS 1149 clinical manifestations that

1147

Pseudohypoparathyroidism, Albright’s Hereditary Osteodystrophy, and Progressive Osseous

Heteroplasia: Disorders Caused by Inactivating GNAS

Mutations*

Murat Bastepe and Harald Jüppner

*Chapter titles shaded in green indicate chapters dedicated predominantly to pediatric endocrinology content.

c h a p t e r 66

C H A P T E R O U T L I N E

DIAGNOSIS AND PROGRESSION OF PTH RESISTANCE, 1148PSEUDOHYPOPARATHYROIDISM TYPE 1a, 1148THE COMPLEX GNAS LOCUS, 1151CLINICALLY DISTINCT, GENETICALLY RELATED PHP 1a VARIANTS, 1153Pseudopseudohypoparathyroidism, 1153Progressive Osseous Heteroplasia, 1154

PSEUDOHYPOPARATHYROIDISM TYPE 1b, 1155PSEUDOHYPOPARATHYROIDISM TYPE 2, 1157ACRODYSOSTOSIS WITH HORMONAL RESISTANCE, 1158TREATMENT, 1158SUMMARY, 1159

In 1942, Albright and colleagues1 described a group of patients who displayed certain physical features, including obesity, short stature, brachydactyly, and cognitive impairment, combined with hypocalcemia and hyperphosphatemia. In these patients, exoge-nous, biologically active parathyroid hormone (PTH) extracts failed to result in a full phosphaturic response; hence, the term pseudohypoparathyroidism (PHP) was introduced, indicating that hypocalcemia and

hyperphosphatemia in these patients resulted from target-organ resistance to, rather than deficiency of, PTH. Consistent with resistance to the actions of this hormone, it was later shown that patients affected by PHP have elevated concentrations of immunoreac-tive PTH.2 Subsequently, it was also shown that some patients affected by PHP have resistance toward other hormones; however, PTH resistance is the most promi-nent feature of the disease.

K E Y P O I N T S

• GNAS is a complex imprinted gene giving rise to Gsα and several other coding and noncoding transcripts.

• Inactivating mutations of GNAS that affect Gsα lead to Albright’s hereditary osteodystrophy, as well as resistance to PTH and other hormones.

• Tissue-specific silencing of the paternal Gsα allele plays an important role in the genetic mechanisms underlying hormone resistance.

• Defects downstream of Gsα in the cAMP signaling pathway can also lead to hormone resistance and skeletal defects similar to those seen in Albright’s hereditary osteodystrophy.

1148 PART 6 PARATHYROID GLAND, CALCIOTROPIC HORMONES, AND BONE METABOLISM

The primary site of PTH resistance in PHP is the renal proximal tubule, as the actions of PTH in bone and the distal tubule appear normal.3-5 Patients with PHP have reduced serum concentrations of 1,25-dihydroxyvitamin D [1,25(OH)2D], which is the main cause of hypocal-cemia.6-9 Both low serum 1,25(OH)2D and hyperphos-phatemia are the direct results of PTH resistance at the proximal tubule. Hyperphosphatemia is typically wors-ened by the elevation of PTH in the circulation and the absence of resistance to the bone resorptive actions of this hormone; on the other hand, the increase in serum PTH can prevent symptomatic hypocalcemia in some PHP patients due largely to its unimpaired actions on the bone and the renal distal tubule,10-14 and thus calcium release from bone and enhanced calcium reabsorption, respectively. However, at some point in their lives, most patients manifest hypocalcemia and, therefore, present with associated clinical findings.

DIAGNOSIS AND PROGRESSION OF PTH RESISTANCEPTH exerts its actions by binding to a seven-transmem-brane, G protein–coupled receptor (the PTH/PTH-related protein receptor, PTHR1)15. Although the PTHR1 can couple to several different G proteins,16 most PTH actions are mediated primarily through the stimulatory G protein, which acts on adenylyl cyclases, thereby increasing the for-mation of intracellular second-messenger cyclic adenosine monophosphate (cAMP).15,17 PTH-induced cAMP forma-tion is used as an important indicator of renal tubular PTH function, since most PHP patients display an inadequate or absent increase of urinary cAMP in response to exogenous, biologically active PTH (Fig. 66-1).18 In fact, the nephrog-enous cAMP response to the exogenously administered PTH is utilized as a test for establishing the diagnosis of PHP, which is more sensitive than measuring the increase in urinary phosphate excretion used in the original Ells-worth-Howard test. However, currently used high-sensi-tivity PTH assays often suffice to make the diagnosis when serum PTH is elevated in the presence of hypocalcemia and hyperphosphatemia. Nonetheless, depending on the nature of the nephrogenous response to exogenous PTH, PHP is subdivided into two main types. PHP type 1 is defined by blunted urinary excretion of both cAMP and phosphate, and PHP type 2 is defined by blunted urinary excretion of phosphate only.1,18,19

Signs and symptoms of decreased serum calcium level often reflect secondary prolongation of the QT interval on EKG and increased neuromuscular excitability lead-ing to Trousseau’s and Chvostek’s signs as well as bron-chospasm. Although the most common manifestations of hypocalcemia include muscle tetany and spasms, find-ings vary markedly among patients. In more severe cases, patients present with seizures. Other neurologic symp-toms can also arise from hypocalcemia, and some patients with PHP have initially been diagnosed with movement disorders.20-24 In one report, two sisters with PHP type 1b (see later) presented with paroxysmal kinesigenic choreoathetosis, and the diagnosis of PTH resistance in one sister was made after approximately 4 years of oral antiepileptic treatment when biochemical evaluation and

genetic testing was performed.25 Some patients present-ing with seizures demonstrate epileptiform EEG changes and, because this activity typically responds to antiepilep-tic treatment, the diagnosis of PHP can be delayed.26,27 As another complication of the changes in serum calcium and phosphorous, brain imaging studies frequently show intracranial calcifications in PHP patients.20,28-34

PHP is a congenital disorder, but only few reports describe findings consistent with PTH resistance during the neonatal period.35,36 Clinical manifestation of hypo-calcemia typically occurs only later in childhood, suggest-ing that PTH resistance and the resultant changes in serum calcium and phosphorous develop only gradually.37-40 In a longitudinal study of a child with PHP 1a (see later), cAMP response to PTH was found to be normal at 3 months of age, whereas it was blunted at 2.6 years of age.38 In another PHP 1a case, a gradual decline of serum calcium, preceded by increasing serum phosphorous and PTH levels, was demonstrated.37 In addition, a PHP 1b patient diagnosed by genetic analysis at birth (see later) was shown to have normal serum PTH levels until 18 months of age, when an elevation of PTH was first detected despite normal serum calcium and phosphorous.39 It thus appears that PTH responses are intact during the early postnatal period despite the existence of the molecular defect underlying PHP. The mechanisms that allow normal PTH signaling during this developmental stage remain unknown.

PSEUDOHYPOPARATHYROIDISM TYPE 1aOf the two main PHP types, PHP type 1 is much more common. Various clinical variants of PHP type 1 have been described based on the presence or absence of

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Figure 66-1 Time-course of urinary cAMP levels in response to PTH infusion. Upon injection of PTH, normal subjects (blue triangles) ex-hibit a 50- to 100-fold peak over the basal, while patients with PHP 1a (open green circles) or PHP 1b (solid red circles) demonstrate a blunted response. (Adapted from Levine MA, Jap TS, Mauseth RS, et al. Activ-ity of the stimulatory guanine nucleotide-binding protein is reduced in erythrocytes from patients with pseudohypoparathyroidism and pseudop-seudohypoparathyroidism: Biochemical, endocrine, and genetic analysis of Albright’s hereditary osteodystrophy in six kindreds. J Clin Endocrinol Metab 62:497-502, 1986).

114966 PSEUDOHYPOPARATHYROIDISM, ALBRIGHT’S HEREDITARY OSTEODYSTROPHY, AND PROGRESSIVE OSSEOUS

clinical manifestations that coexist with PTH resistance, diminished stimulatory G protein activity in easily acces-sible cells, and silenced expression of the GNAS-derived α-subunit of the stimulatory G protein (Gsα) (Table 66-1).

As described originally by Albright and colleagues,1 PHP patients often exhibit characteristic physical fea-tures that may include obesity, round facies, short stature, brachydactyly, ectopic ossification, and men-tal impairment (Fig. 66-2). These features are termed Albright’s hereditary osteodystrophy (AHO) and occur primarily in PHP patients who are now classified as hav-ing pseudohypoparathyroidism type 1a (PHP 1a). The brachydactyly observed in patients with AHO typically involves the metacarpal and/or metatarsal bones and, thus, the pattern of shortening of the hand bones dif-fers from other disorders with brachydactyly, such as familial brachydactyly and Turner’s syndrome.41 Due to shortened metacarpals, dimpling over the knuckles of a clenched fist (Archibald sign) is often observed.42 The shortening of the distal phalanx of the thumb, however, is the most common skeletal abnormality (called murderer’s thumb or potter’s thumb), and some patients have shortening of all digits.43 Mental impair-ment is mild, often presenting as cognitive defects. It is possible that the cause of mental impairment is the deficiency of Gsα signaling in the brain. While hypocal-cemia and/or hypothyroidism may also play a role in this phenotype, correction of these biochemical defects does not prevent mental impairment in all cases. There is remarkable patient-to-patient variability in AHO, even among patients that carry the same genetic muta-tion and belong to the same family (see later for a dis-cussion of the underlying genetic defect). Some patients may exhibit a single AHO feature only, such as obesity, while others may present with multiple AHO features.

Furthermore, the severity of each feature differs vastly among the patients. In addition, individual AHO fea-tures are not unique to PHP, as they can be observed in other unrelated disorders, for example, acrodysos-tosis caused by PDE4D mutations44,45 or brachydac-tyly–mental retardation syndrome caused by HDAC4 mutations.46 The variable expressivity and the lack of specificity of individual features can make the AHO diagnosis challenging. While the coexistence of hor-mone resistance in PHP 1a patients is often helpful, it can also be misleading. This is particularly important for the differential diagnosis of different PHP forms characterized by the presence of AHO features alone or hormone resistance alone (see later).

In addition to having PTH resistance and AHO, patients with PHP 1a show clinical evidence that is consis-tent with target-organ resistance to other hormones. The most common additional hormone resistance involves the actions of TSH, leading to hypothyroidism.47,48 In fact,

TABLE 66-1 Clinical and Molecular Features of the Different PHP Forms and ADOHR

PTH Resistance

Additional Hormone Resistance

Typical AHO Features Genetic Defect

PHP 1a/1c

Yes Yes Yes Coding GNAS mutations

PPHP No No Yes Coding GNAS mutations

POH* No No No Coding GNAS mutations

PHP 1b Yes Some cases

No† Microdeletions affecting GNAS imprinting , patUPD20q, or unknown defects.

PHP 2 Yes No No UnknownADOHR Yes Yes Yes Coding PRKAR1A

mutations

ADOHR, Acrodysostosis with hormonal resistance; AHO, Albright’s hereditary osteodystrophy; PHP, pseudohypoparathyroidism; POH, progressive osseous heteroplasia; PTH, parathyroid hormone.

*Note that POH-like severe heterotopic ossifications have been reported in some patients with multiple hormone resistance.

†Some recent studies have demonstrated coexistence of AHO features and GNAS imprinting defects.

B

A

C

Figure 66-2 Albright’s hereditary osteodystrophy (AHO). A, Short stature and obesity are among the typical features of AHO. B, Hand radiograph of a child with AHO demonstrating short fourth and fifth metacarpals at 5 11/12 years of age. C, The same patient’s hand radio-graph at 8 10/12 years of age. (A, Adapted from Albright F, Burnett CH, Smith PH, Parson W. Pseudohypoparathyroidism—an example of “Seabright-Bantam syndrome.” Endocrinology 30:922-932, 1942.)


unlike PTH resistance, which typically develops later in life, TSH resistance can be present at birth.49-52 Resis-tance to gonadotropins and growth hormone–releasing factor has been reported,53-55 whereas resistance to other peptide hormones that also mediate their actions through Gsα-coupled receptors, such as vasopressin or ACTH, does not appear to become clinically overt.54,56-60

The genetic mutation that causes PHP 1a is located within the Gsα-coding GNAS exons.61,62 A protein that is essential for the actions of many hormones, Gsα primar-ily mediates agonist-induced generation of intracellular cAMP. Activation of a stimulatory G protein–coupled receptor by its agonist, such as PTH, leads to a GDP-GTP exchange on Gsα, causing dissociation of the latter from Gβγ subunits (Fig. 66-3). This allows both Gsα and Gβγ to stimulate their respective effectors. In its GTP-bound state, Gsα can directly activate several different effectors, such as Src tyrosine kinase,63 and certain Ca-channels.64,65 Apart from these effectors, however, ade-nylyl cyclase is by far the most ubiquitous and the most extensively investigated effector molecule stimulated by Gsα. An integral membrane protein, adenylyl cyclase cat-alyzes the synthesis of the ubiquitous cAMP, which then triggers various cell-specific responses. The activation of adenylyl cyclase and other effectors by Gsα is regulated by the intrinsic GTP hydrolase (GTPase) activity of Gsα. Conversion of GTP into GDP results in the re-assembly of the G protein heterotrimer and, thereby, prevents further effector stimulation (see Fig. 66-3).

Mutations identified in PHP 1a patients are heterozy-gous and scattered throughout all of the 13 GNAS exons encoding Gsα and the intervening sequences, including missense and nonsense amino acid changes, insertions/deletions that cause frameshift, and nucleotide alterations

that disrupt pre-mRNA splicing (an extensive list of these mutations can be found online at OMIM entry #139320 at http://www.ncbi.nlm.nih.gov). Constitutional dele-tions of the chromosomal arm containing GNAS have also been identified.66 Consistent with this mutational spectrum, Gsα level/activity is reduced by approximately 50% in easily accessible tissues from PHP 1a patients, such as erythrocytes, skin fibroblasts, and platelets.48,67-80 Deficiency of Gsα has also been demonstrated in renal membranes from a patient with PHP.81 A complementa-tion assay is typically used for examining Gsα activity, involving patient-derived erythrocyte membranes and membranes from turkey erythrocyte that lack endogenous Gsα (Fig. 66-4). Gsα hypofunction can also be detected in patient-derived platelets by using a functional test that examines the ability of prostaglandin E1 or prostacyclin to inhibit platelet aggregation.82 The detection of reduced Gsα activity is important for the establishment of PHP 1a diagnosis, particularly for cases in which genetic analysis fails to reveal a GNAS mutation. Reduction of Gsα activ-ity in PHP 1a is consistent with the fact that PTH and the other hormones with impaired actions in this disorder act via cAMP-mediated signaling pathways.

Among the many different inactivating GNAS muta-tions, a 4-bp deletion in exon 7 has been identified in numerous families, representing a genetic “hot-spot.” In addition, two different mutations are associated with additional phenotypes. A missense mutation in exon 13 (A366S) was identified in two unrelated boys, who pre-sented with PHP 1a and precocious puberty.83,84 This mutant Gsα protein is temperature-sensitive and thus rap-idly degraded at normal body temperature. The amino acid substitution, however, renders the protein constitutively active, resulting in elevated cAMP signaling in the cooler temperature of the testis. More recently, another mutant Gsα protein has been described in a unique case of familial PHP 1a and transient neonatal diarrhea.85 The mutation, which entails a repeated AVDT sequence at residues 366 to 369, generates an unstable but constitutively active Gsα

βγ

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Figure 66-3 Heterotrimeric G protein activation cycle. The heterotri-meric complex is assembled at the basal state, with the α-subunit being associated with a GDP molecule. Upon binding of an agonist (A) to its Gs-coupled receptor (R), the GDP molecule bound to the α-subunit is replaced with a GTP molecule, that is, the activated receptor acts as a guanine nucleotide exchange factor for the α-subunit. The GTP-bound form of the α-subunit dissociates from the βγ subunits and thereby stimulates its downstream effectors, which, in the case of Gsα, includes adenylyl cyclase. Note that the free Gβγ dimer can also stimulate differ-ent downstream effectors. The intrinsic GTP hydrolase activity of the α-subunit converts the GTP into GDP, resulting in the re-association of the heterotrimer and, therefore, termination of effector stimulation.

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Figure 66-4 Gsα erythrocyte activity in PHP 1a, PPHP, and PHP 1b patients. The measurement is conducted by complementation of patient-derived erythrocyte membranes with membranes from S49cyc– lymphoma cells, which genetically lack Gsα but retain all other com-ponents necessary for hormone-activated adenylyl cyclase response. (Adapted from Levine MA. Hypoparathyroidism and pseudohypopara-thyroidism. In De Groot LJ, Jameson J, eds. Endocrinology, 4th ed. Philadelphia: Saunders, 2000, pp. 1133-1153.).

http://www.ncbi.nlm.nih.gov


mutant due to enhanced GDP-GTP exchange and reduced GTPase activity. While hormone resistance results from the instability of the Gsα-AVDT mutant, the diarrhea is attributed to increased plasma membrane localization of the mutant protein in the small intestine epithelium. Another pediatric case has been described in whom a de novo missense mutation (R231C) on the paternal allele was found together with a maternally inherited combina-tion of three C-to-T substitutions, resulting in aberrant GNAS splicing.86 The patient with these compound het-erozygous mutations had morbid obesity, TSH and PTH resistance, and a prothrombotic state due to marked Gsα hypofunction in platelets.

PHP 1c has been described as a distinct variant of PHP 1a,70, but the clinical and laboratory features of patients with PHP 1c are identical to those with PHP 1a, as they have both AHO and multihormone resistance. In contrast to PHP 1a, however, biochemical assays demonstrate no reduction in Gsα activity in erythrocytes obtained from PHP 1c patients, suggesting the absence of mutations within the Gsα gene. Nevertheless, recent molecular characterizations have revealed Gsα mutations at least in some PHP 1c patients. However, the Gsα mutants show only impaired coupling to different G protein–coupled receptors, yet show normal Gsα activity in complementa-tion assays that use non-hydrolyzable GTP analogues for stimulation of Gsα activity rather than a receptor ago-nist.43,87 Thus, the mutant Gsα protein causing PHP 1c

allows basal cAMP formation but is unable to trigger an increase in response to hormones. Hence, at least some PHP 1c cases are allelic variants of PHP 1a. Although it remains possible that some patients who match the description of PHP 1c develop hormone resistance and AHO due to a novel gene mutation that affects cAMP production without functionally impairing Gsα itself (e.g., inactivating mutations affecting one of the adeny-lyl cyclases or activating mutations in the phosphodies-terase), it appears unlikely that such putative mutations affect only few G protein–coupled receptors and thus cause limited hormonal resistance as observed in PHP 1a and PHP 1c, but instead dictate the phenotype by the tissue-specificity of their expression levels.

THE COMPLEX GNAS LOCUSGNAS is a complex locus giving rise to multiple different coding and noncoding transcripts that show monoallelic, parent-of-origin–specific expression profiles (Fig. 66-5). GNAS maps to the telomeric end of the long arm of chro-mosome 20 (20q13.2-20q13.3).88-90 Gsα is encoded by 13 exons,91 but due to alternative pre-mRNA splicing, the Gsα transcript has several variants. The long and the short Gsα variants (Gsα-L and Gsα-S, respectively) differ from each other by the inclusion or exclusion of exon 3;91-93 these Gsα variants are typically detected as 52- and 45-kD protein bands on Western blots. Showing further

CH3 – – – Paternal

MaternalCH3CH3CH3–

5 NESP554 3 2 1 XL A20 A21 A/B 1 2 3 N1 4 5 6 7 13

*exons 2–13

*exons 2–13

XL

AS exons 1–5

AS

*exons 2–13

exons 2–13

NESP55

Maternal

Paternal

Biallelic(most tissues)

*

exons 1

A/B

Figure 66-5 The GNAS locus. The complex GNAS locus gives rise to multiple transcripts. Boxes and connecting lines indicate exons and introns, respectively. Arrows show the direction of transcription for each transcript. The main transcriptions derived from GNAS and the utilized exons are depicted as rectangles below the gene schematic. Gsα is biallelically expressed except in a small number of tissues, including renal proximal tubules, thyroid glands, gonads, and pituitary gland, in which expression from the paternal allele is silenced (dashed arrow). XLαs, A/B and antisense (AS) transcripts are derived from the paternal allele, and the NESP55 transcript from the maternal allele. Promoters of XLαs, A/B, antisense, and NESP55 transcripts are methylated on the silenced allele, as indicated by CH3 (methylated CpGs) and - (unmethylated CpGs).


complexity, each Gsα form either includes or excludes a CAG trinucleotide (encoding serine) at the start of exon 4. Small, but potentially important, differences have been reported between the activities of Gsα-L and Gsα-S. For example, Gsα-L has been shown to display greater abil-ity to mediate receptor signaling than Gsα-S when par-tially purified proteins from rabbit liver were examined,94 although the opposite finding was reported upon the use of cultured pancreatic islet cells.95 Furthermore, the GDP release rate from Gsα-L appears to be approximately twofold higher than Gsα-S,96 and, accordingly, fusion proteins involving the β2-adrenergic receptor and Gsα-L have shown higher constitutive activity than those involv-ing the receptor and Gsα-S.97 Moreover, differences in the subcellular targeting of these two Gsα variants have been reported.98-100 Currently, it remains unclear whether these differences translate into biologically significant effects, such as divergence in the variety of effectors and/or the efficiency of effector activation. Nonetheless, a single nucleotide insertion in exon 3 leading to frameshift and early termination has been recently identified in two siblings affected by a mild form of PHP 1a.101 The mild-ness of the phenotype is consistent with generation of one of the two main Gsα variants (i.e., Gsα-S). It remains unknown whether this exon 3 mutation impairs agonist responses in an effector- and tissue-specific manner, and this possibility depends on the putative effector selectivity and relative expression levels of Gsα-L and Gsα-S in dif-ferent tissues.

Recent studies have revealed that GNAS gives rise to multiple additional gene products that show parent-of-origin–specific, monoallelic expression. Besides Gsα, at least two translated GNAS transcripts exist, using distinct upstream promoters and alternative first exons that splice onto exons 2 to 13 encoding Gsα. The most upstream promoter relative to the Gsα promoter drives expression of a neuroendocrine secretory protein with an apparent molecular mass of 55,000 (NESP55).102 The paternal NESP55 promoter is methylated, and the transcription occurs from the maternal allele.103,104 In humans, NESP55 protein is encoded by a single exon, so that Gsα exons 2 to 13 compose the 3′ untranslated region.103,104 Expressed in neuroendocrine tissues, peripheral and central nervous system, and some endocrine tissues,102,105-108 NESP55 is a chromogranin-like protein associated with the con-stitutive secretory pathway.109 Loss of the expression of NESP55 protein does not seem to have an overt clinical outcome in humans, as evidenced from patients with PHP type 1b (see later). However, its disruption in mice result in a subtle behavioral phenotype characterized by increased reactivity to novel environments.110 Transcrip-tion from the NESP55 promoter is likely to play a role in the regulation of GNAS imprinting (see later).

Another GNAS product is XLαs, which is expressed from the paternal allele.104,111,112 XLαs is expressed abun-dantly in various parts of the brain and neuroendocrine tissues, and its expression can be readily detected in a variety of other fetal and postnatal tissues.111,113-120 XLαs also uses a distinct upstream promoter and a unique first exon that splices onto Gsα exons 2 to 13.104,112 Unlike in the NESP55 transcript, however, the latter portion of

the XLαs transcript is included in the translated product, resulting in a protein that is partially identical to Gsα.111 Consistent with its structural similarity to Gsα, XLαs can mediate receptor-activated adenylyl cyclase stimulation in transfected cells and in transgenic mice.43,115,121,122 In fact, it appears to be basally more potent than Gsα when expressed at levels comparable to the latter and is able to prolong PTH signaling in transfected cells.120,123 How-ever, the phenotype of mice with targeted ablation of XLαs suggests that XLαs has unique, albeit as-yet-undefined, cellular functions. These mice show high early postnatal mortality due to poor adaptation to feeding and impair-ment in the glucose and energy metabolism,118 in contrast to Gsα knockout mice, which to a large extent recapitu-late the findings in patients with PHP 1a.124,125 Findings from adult mice with disrupted XLαs expression indicate that this protein is a negative regulator of increased sym-pathetic nervous system activity in mice.126,127

The paternal GNAS also gives rise to two additional transcripts. From the sense strand originates the A/B tran-script (also termed 1A or 1′), which, similar to NESP55 and XLαs, utilizes an upstream promoter and an alter-native first exon (exon A/B) that splices onto Gsα exons 2 to 13.128,129 Exon A/B does not comprise an in-frame translation initiation codon but, as demonstrated in vitro, the translation can be started through the use of an AUG located within exon 2, thereby giving rise to an N-termi-nally truncated protein that localizes to the plasma mem-brane when transiently expressed in cell lines.129 Until recently, no evidence has supported the existence of an endogenous A/B protein, and therefore, it was thought that the A/B transcript was a noncoding RNA. Endo-genous A/B protein has recently been detected, however, in human fetal kidney by Western blot using an antibody against the C-terminal end of Gsα.130 It remains possible that A/B has functions both as a protein and as a tran-script regulating the expression within the GNAS locus (see later). Another paternal GNAS transcript is derived from the antisense strand.131,132 The GNAS antisense transcript (GNAS-AS1), which is formed in humans by at least six primary exons that show multiple alternative splicing patterns,131,133 is also considered to be noncod-ing. Data from mouse models indicate that GNAS-AS1 plays a critical role in silencing the paternal NESP55 pro-moter in cis.134 The promoter of GNAS antisense tran-script is immediately upstream of the promoter of XLαs. Although the promoters of XLαs and antisense tran-script are located together within a large differentially methylated region (DMR), the female germ line–specific imprint is established at the antisense promoter only.135 The A/B promoter is likewise methylated in the female germ line, but not in the male germ line.136 Thus, the two germ line imprint marks at the GNAS locus include the promoters of the antisense and A/B transcripts. Accord-ingly, data from different genetically manipulated mouse models show that these noncoding transcripts are essen-tial for the regulation of imprinted gene expression from GNAS.134,137,138 Imprinted A/B transcription (i.e., expression from only the non-methylated maternal allele) is particularly important for the development of hormone resistance seen in patients with PHP 1b (see later).


Unlike the promoters of NESP55, antisense, XLαs, and A/B transcripts, the promoter of Gsα is not differentially methylated and, accordingly, Gsα expression is biallelic in most tissues.103,112,139,140 Biallelic Gsα expression has been specifically shown in human bone and adipose tis-sue.141 However, paternal Gsα expression is silenced in a small subset of tissues through as-yet-undefined mecha-nisms, so that the maternal allele is the predominant source of Gsα expression. These tissues include the renal proximal tubule, thyroid, pituitary, and gonads.125,142-144 Although devoid of differential methylation, the Gsα pro-moter exhibits parent-of-origin–specific histone modifica-tions in those tissues where it is monoallelic. The active maternal Gsα promoter shows a greater ratio of tri- to di-methylated histone-3 Lys4 compared to the silenced paternal promoter in the proximal tubule, whereas the amount of methylated histones is similar in maternal and paternal Gsα promoters in liver, a tissue in which Gsα is biallelic.145 As discussed later, the tissue-specific, paternal Gsα silencing has a key role in the development of PTH resistance in patients with PHP 1a and PHP 1b.

CLINICALLY DISTINCT, GENETICALLY RELATED PHP 1a VARIANTS

PseudopseudohypoparathyroidismPhysical abnormalities similar to those observed in patients with PHP 1a but without evidence for an abnormal regulation of calcium and phosphate homeostasis were first reported in 1952.146 Because of the lack of an abnor-mal regulation of mineral ion homeostasis, the name pseudopseudohypoparathyroidism (PPHP) was coined to describe this disorder.146 Interestingly, patients with PPHP also carry GNAS mutations that lead to dimin-ished Gsα function, and these mutations can be found in the same kindred as those with PHP 1a. However, both disorders are never seen in the same sibling kinship, and a careful analysis of multiple families has revealed that the mode of inheritance of each disorder depends on the gender of the parent transmitting the Gsα mutations.147 Thus, an inactivating Gsα mutation causes PHP 1a (i.e., hormone resistance and AHO) after maternal inheritance, whereas the same mutation on the paternal allele results in PPHP (AHO only). Most AHO features, except obesity and mental retardation, appear to develop regardless of the parent of origin, and it is therefore primarily hormone resistance that displays an imprinted mode of inheritance. Recent studies have furthermore revealed that most PPHP patients are considerably smaller at birth, particularly if their inactivating Gsα mutation is located in GNAS exons 2 to 13 of the paternal allele.148-150

The tissue-specific imprinting of Gsα expression can explain the parent-of-origin–specific inheritance of hor-mone resistance. In those tissues where Gsα expression is paternally silenced (i.e., Gsα is expressed exclusively or predominantly from the maternal allele), an inactivating mutation located on the paternal allele is not predicted to alter Gsα function, whereas the same mutation located on the maternal allele is predicted to abolish Gsα func-tion completely (Fig. 66-6). The tissue-specific imprint-ing of Gsα expression also explains why the target organ

resistance involves only a small subset of tissues despite the involvement of Gsα signaling in a multitude of physi-ologic responses. Hormone resistance is observed in those tissues where Gsα is imprinted, such as the proximal renal tubule and the thyroid gland, while hormone responses are unimpaired in those tissues where Gsα is biallelic, such as the distal renal tubules. The role of tissue-specific Gsα imprinting in the development of PTH resistance has been demonstrated through the generation of mice heterozy-gous for maternal or paternal disruption of GNAS.142 A recent study furthermore showed that the silencing of the paternal Gsα allele in the renal proximal tubule develops after the early postnatal period in mice, thus providing a plausible explanation for the finding that the manifes-tation of PTH resistance occurs mostly after infancy in patients with PHP 1a and PHP 1b.151

The finding that most AHO features develop regardless of the parent transmitting a Gsα mutation has led to the hypothesis that the inheritance of AHO is due to Gsα hap-loinsufficiency in various tissues, which appears to be true in certain settings. PTHrP-induced cAMP generation is critical for proper control of hypertrophic differentiation of growth plate chondrocytes,152 and Gsα haploinsuffi-ciency has been demonstrated in this tissue through the study of mice chimeric for wild-type cells and mutant cells heterozygous for disruption of GNAS exon 2.153 Regard-less of the parental origin of the GNAS exon 2 disruption, the mutant cells displayed premature hypertrophy com-pared to their wild-type neighbors, although the paternal disruption (i.e., loss of one Gsα allele combined with a complete loss of XLαs) resulted in significantly more pre-mature hypertrophy than the maternal disruption (loss of one Gsα allele only). Thus, the brachydactyly and/or short stature observed in the context of AHO likely result from diminished Gsα signaling in growth plate chondrocytes. While these data correlate well with the notion that AHO develops after both maternal and paternal inheritance of a Gsα mutation, recent evidence suggests that individual AHO features can also be subject to imprinting. A care-ful analysis of multiple patients with PHP 1a and PPHP

hormone resistance andAHO, i.e., PHP 1a

p

m

p

m AHO only, i.e., PPHP

Figure 66-6 The effect of paternal Gsα silencing in the development of hormone resistance. Gsα is biallelic in most tissues, and a heterozy-gous inactivating Gsα mutation therefore causes an approximately 50% reduction in Gsα activity/expression regardless of the parent of origin of the mutation. However, in some tissues, such as the proximal tubule and thyroid gland, paternally inherited Gsα transcript is silenced (X). Thus, if a Gsα mutation (black square) is inherited from a female indi-vidual, this mutation nearly abolishes the expression or the activity of Gsα in those tissues, thus leading to hormone resistance (PHP 1a). In contrast, upon paternal inheritance, the same mutation Gsα does not lead to a significant change in the expression/activity, and thus, the hor-mone responses are unimpaired. Paternal and maternal Gsα alleles are depicted by white or grey rectangles.


revealed that obesity is primarily a feature of PHP 1a patients, developing after maternal inheritance.154 Con-sidering that Gsα is biallelic in the white adipose tissue,141 it was proposed that Gsα may also be imprinted (pre-dominantly maternal expression) in areas of the central nervous system that control satiety and body weight.154 A recent report has also demonstrated that cognitive impairment is more prevalent in PHP 1a than in PPHP, thus indicating that tissue-specific Gsα imprinting may involve additional brain regions.155 On the other hand, imprinted inheritance has not been reported regarding short stature, despite the finding that Gsα is maternally expressed in the pituitary gland143,144 and that PHP 1a patients display GHRH resistance that results in growth hormone (GH) deficiency.54,55 Conversely, as outlined ear-lier, the small for gestational age phenotype is associated more strongly with Gsα mutations on the paternal allele than with Gsα mutations on the maternal allele.148,150 A single study also showed that PPHP patients carrying mutations within exons 2 to 13 are born smaller than those carrying mutations within exon 1,150 thus impli-cating both Gsα haploinsufficiency and the deficiency of a paternally expressed GNAS product, such as XLαs, in the pathogenesis. Future analyses of patients with PHP 1a and PPHP will be helpful in determining the relative roles of genomic imprinting and haploinsufficiency in the development of individual AHO features.

Progressive Osseous HeteroplasiaA disorder termed progressive osseous heteropla-sia (POH) has been described in patients with severe

extraskeletal ossifications that involve deep connective tissue and skeletal muscle156,157 (Fig. 66-7). In POH, the ectopic bone is primarily intramembranous, as opposed to a similar disease termed fibrodysplasia ossificans pro-gressiva (FOP) in which extraskeletal bone formation occurs via endochondral mechanisms, and is accompa-nied by skeletal malformations.158,159 Few patients with POH demonstrate AHO features and, consistent with the occasional coexistence of these two sets of clinical defects, heterozygous inactivating Gsα mutations have been identified as a cause of POH.160-162 Several of the identified mutations are identical to those found in PHP 1a/PPHP kindreds.161,162 Gsα activity and downstream signaling has been implicated in the control of osteo-genic differentiation. Patients who are mosaic for hetero-zygous GNAS mutations that result in constitutive Gsα activity develop fibrous dysplasia of bone characterized by irregular woven bone disrupted by fibrous tissue.163 Moreover, in human mesenchymal stem cells, reduction of Gsα protein levels has been shown to cause osteo-genic differentiation, while inhibiting the formation of adipocytes.164,165 In addition, Runx2, a key regulator of osteoblast-specific gene expression, appears to sup-press Gsα expression.166 Thus, osteoprogenitor forma-tion and the early stages of osteoblastic differentiation require reduced levels of cAMP signaling, consistent with the association of inactivating Gsα mutations with the severe ectopic bone formation observed in POH.

Because of the presence of GNAS mutations in both AHO and POH, it appears that additional factors, such as genetic background, epigenetic events, or environmental

Figure 66-7 Clinical and radio-graphic appearance of progressive osseous heteroplasia. A, Posterior view of the legs and feet of a 5-year-old girl with POH showing severe maculopapular lesions caused by extensive dermal and subcutaneous ossification. B, Lateral radiogram of the right leg of an 11-year-old girl with POH demonstrating se-vere heterotopic ossification of the soft tissues. C, Computed tomo-graphic image of the thighs of a 10-year-old boy with POH show-ing atrophied soft tissues in the left leg and extensive ossification of the skin, subcutaneous fat, and quadricep muscles. (Adapted from Shore EM, Ahn J, Jan de Beur S, et al. Paternally inherited inactivat-ing mutations of the GNAS1 gene in progressive osseous heteroplasia. N Engl J Med 346:99-106, 2002).

A

B

C


factors may determine the penetrance and severity of the ectopic ossifications in these patients that show approxi-mately 50% loss of Gsα activity. Nevertheless, clinical and genetic data demonstrate several important differ-ences between patients with AHO and those with POH. First, the ectopic bone in AHO is limited to subcutane-ous tissue. In addition, in nearly all patients with POH, the severe ectopic bone formation is isolated (i.e., other typical AHO features are not manifest).162,167 Moreover, mutations leading to isolated POH are inherited from male obligate gene carriers only (i.e., inheritance pattern is exclusively paternal).162 In fact, in one large kindred, paternal inheritance of a GNAS mutation caused POH, while maternal inheritance of the same mutation caused typical AHO findings (without severe heterotopic ossifi-cation).162 These findings indicate that the disease mecha-nism underlying POH is significantly different from that underlying AHO and that deficiency of a GNAS product with exclusive paternal expression, such as XLαs, contrib-utes to the molecular pathogenesis of POH. In addition, a recent study analyzing 12 patients with POH has revealed that the lesions follow a predominantly dermomyotomal distribution that often shows a lateralization bias.168 Based on these findings, which redefine the clinical defini-tion of this disorder, it was hypothesized that the patho-genesis involves progenitor cells of somitic origin, which may undergo loss of heterozygosity at the GNAS locus and thereby cause severe or complete Gsα deficiency.168

PSEUDOHYPOPARATHYROIDISM TYPE 1bAnother form of PHP was described by Peterman and Garvey169 and by Reynolds and associates.170 Now known as pseudohypoparathyroidism type 1b (PHP 1b), this PHP form is characterized by the presence of PTH-resistant hypocalcemia and hyperphosphatemia, but without evidence of AHO in most cases. In addition to increased serum PTH, patients with PHP 1b can dem-onstrate elevated serum alkaline phosphatase activity, which suggests normal PTH-dependent bone turnover.171 In fact, hyperparathyroid bone disease can be observed in association with PHP 1b, especially in patients with sporadic PHP 1b or in the index cases of the autosomal dominant form of PHP 1b (AD-PHP 1b). This occurs less frequently, however, in PHP 1a since this variant is associated with AHO features and is therefore diagnosed earlier in life. The intact PTH response in the bone is con-sistent with the lack of Gsα imprinting in bone141 and led to the introduction of the term pseudohypo-hyperpara-thyroidism (PHP-HPT).172-174

The hormone resistance observed in PHP 1b patients develops only after maternal inheritance of the genetic defect175 (i.e., the mode of inheritance is identical to the hormone resistance in PHP 1a). PTH resistance and related changes in calcium and phosphate homeostasis are the major laboratory findings in PHP 1b, but some PHP 1b patients also display mild hypothyroidism with slightly elevated TSH levels26,176-178 as well as some eleva-tion in calcitonin level.176 Hypothyroidism, as in patients with PHP 1a, likely results from mild TSH resistance and is consistent with the predominantly maternal Gsα

expression in the thyroid gland.144,177 Nevertheless, evi-dence for resistance to other hormones, such as gonado-tropins, whose actions also involve tissues in which Gsα is imprinted, has not been reported for PHP 1b patients. A study assessed growth hormone response to GHRH plus arginine stimulation in PHP 1b, revealing a normal response in 9 of 10 patients.178 On the other hand, in addition to PTH and mild TSH resistance, hypourice-mia due to increased fractional excretion of uric acid has been reported in the affected individuals of two unrelated PHP 1b kindreds.27,179 This finding implicates impaired PTH actions in the development of hypouricemia in these patients, an interpretation that is consistent with two previous reports describing hyperuricemia in association with hyperparathyroidism.180,181 However, hypouricemia resolved in one of the PHP 1b kindreds following treat-ment with calcium and calcitriol.27

Patients with PHP 1b display normal Gsα bioactivity/levels in easily accessible tissues. Accordingly, coding Gsα mutations are ruled out in these patients. In one family, however, a mutation located in exon 13 (in-frame deletion of Ile,382 del382Ile) was reported, leading to the uncoup-ling of Gsα from the PTHR-1 but not other receptors that were expressed in LLCPK cells, including the TSHR, LHR, and β-adrenergic receptor,182 which are of renal origin and express endogenous Gsα. These findings suggested an iso-lated PTH resistance as seen in PHP 1b, leading to the con-clusion that the del382Ile mutation in Gsα represents a rare cause of PHP 1b. However, this conclusion has been ques-tioned, as the use of transfected mouse embryonic fibroblasts null for endogenous Gsα showed that del382Ile leads to uncoupling from not only PTHR-1 but also the β-adrenergic receptor.43 Because of a lack of Gsα mutations and because Gsα activity/levels in easily accessible tissues are normal in PHP 1b patients, inactivating mutations that affect the gene encoding PTHR-1 was considered in the past.183 How-ever, several different studies have excluded such mutations as the cause of this disease.184-187 Instead, homozygous or compound heterozygous, inactivating mutations of PTHR-1 have been revealed as the cause of Blomstrand’s chondro-dysplasia, an embryonic lethal disorder with severe skeletal abnormalities.188 A homozygous PTHR-1 mutation was furthermore identified in patients with Eiken syndrome, an autosomal recessive skeletal dysplasia,189 and heterozygous inactivating PTHR-1 mutations were observed in several families with an autosomal dominant form of delayed tooth eruption190-193 (i.e., disorders without evidence for hypocal-cemia and hyperphosphatemia because of impaired actions of PTH).

Based on genomewide linkage analysis, the genetic defect underlying PHP 1b maps to a region of chromo-some 20q13.3 that comprises the GNAS locus,175 but the critical interval excludes all the coding GNAS exons, including those that encode Gsα.26,175 On the other hand, patients with PHP 1b display epigenetic abnormalities within the GNAS locus.26,194 The most consistent epi-genetic defect is a loss of imprinting at exon A/B (also termed exon 1A), which is primarily found as an iso-lated defect in familial PHP 1b cases.26,194 In addition, many sporadic and some familial PHP 1b cases show additional loss of imprinting at the DMR comprising the


XLαs and antisense promoters and a gain of imprint-ing at the DMR composing exon NESP55.13,194 These abnormalities are associated with biallelic expression of A/B, XLαs, and antisense transcripts and silencing of the NESP55 transcript. Together with the genetic link-age data, these imprinting defects suggested that the mutation causing PHP 1b disrupts a regulatory element that controls GNAS imprinting. However, evidence for incomplete penetrance regarding the GNAS imprinting defects has been reported in one kindred, in whom some individuals lacked loss of imprinting and were healthy despite maternally inheriting the disease-associated hap-lotype.195 Thus, the imprinting abnormalities of GNAS appear to be required for the development of PHP 1b. Consistent with the importance of imprinting in the dis-ease mechanism, several patients with PHP 1b have been reported to have paternal uniparental isodisomy involv-ing whole or parts of chromosome 20 that comprise the GNAS locus.176,196-199

In multiple familial PHP 1b cases with isolated exon A/B loss of imprinting, a unique 3-kb microdeletion at the centromeric neighboring STX16 locus has been identified200 (Fig. 66-8). The deleted region comprises STX16 exons 4 to 6 and is flanked by two direct repeats, which may underlie the mechanism whereby this dele-tion occurs. This is consistent with the presence of the same microdeletion in many unrelated families with dif-ferent ethnic and racial origin.13,25,27,178,179,201,202 In a single kindred, a different microdeletion within STX16 has been reported, removing exons 2 to 4 and overlap-ping with the 3-kb microdeletion by approximately 1.3

kb.39 Recently, a maternally inherited 24.6-kb deletion composing STX16 exons 2 to 8 has also been discov-ered in another family with this disorder.203 Thus, the disruption of STX16 appears to be the common genetic defect in cases with isolated loss of exon A/B imprint-ing. The parental origin of these STX16 deletions cor-relates well with the mode of inheritance of PHP 1b. It is maternally inherited in affected individuals and pater-nally inherited in unaffected carriers. This gene encodes syntaxin-16, a member of the SNARE family proteins. However, STX16 does not appear to be imprinted,39,200 and it is therefore unlikely that the loss of one STX16 copy leads to PHP 1b. Instead, since the maternal inheri-tance is associated with loss of exon A/B imprinting on the same allele, these deletions are presumed to disrupt a cis-acting element controlling the establishment or maintenance of exon A/B imprinting. Other than genetic evidence, however, no currently available data corrobo-rate this prediction. A mouse model carrying a deletion equivalent to the 3-kb STX16 deletion in humans has been generated, but neither maternal nor paternal inher-itance of this genetic alteration causes PTH resistance or any alterations in GNAS imprinting204; animals with the homozygous Stx16 deletion are also healthy. It thus appears plausible that the imprinting control element of GNAS located within STX16 in the human is not pre-cisely at the same location in the mouse. Nonetheless, the absence of a phenotype in the Stx16 deletion mice argues against a model in which syntaxin 16, the prod-uct of this gene, is required in the oocyte for proper exon A/B imprinting.

GNAS

STX16

3 kb

5 4A 4 23 1 XL A/B 1 2–13

19 kb4.4 kb

4.0 kb/4.7 kb

4.2 kb24.6 kb

NESP55

CH3

CH3 CH3 CH3 mat

pat

–

– – –

Figure 66-8 Mutations identified in AD-PHP 1b patients and their effects on GNAS imprinting. The most frequent mutation is a 3-kb deletion within STX16, a gene located more than 200 kb upstream of GNAS. This deletion and another overlapping deletion in the same gene are predicted to disrupt a cis-acting control element of GNAS that is required for the imprint mark located at exon A/B. The same prediction is also true for a 19-kb deletion of NESP55 and upstream genomic regions, which was identified in affected individuals of an AD-PHP 1b kindred with isolated loss of A/B methylation. Deletions of the NESP55 DMR including exons 3 and 4 of the antisense transcript and a more recently identified deletion that only includes antisense transcript exons 3 and 4 have been identified in some AD-PHP 1b kindreds. These reveal a cis-acting element controlling imprinting of the entire maternal GNAS allele. Boxes and connecting lines indicate exons and introns, respectively. STX16 exons and GNAS exons 2 to 13 are shown as single rectangles for simplicity. Paternal (pat) and maternal (mat) methylation (CH3), and parental origin of transcription (arrows) are indi-cated. Tissue-specific silencing of the paternal Gsα transcription is depicted by a dotted arrow. The identified deletions are shown by horizontal bars.


In two unrelated familial cases of PHP 1b in whom the affected individuals carried broad GNAS imprint-ing defects, maternally inherited deletions of the entire NESP55 DMR, including exons 3 and 4 of the antisense transcript, have been identified (see Fig. 66-8).133 The deletions are 4 kb and 4.7 kb large and have breakpoints located in similar locations. The unaffected carriers in these families display an apparent loss of NESP55 methy-lation due to the loss of this region from the normally methylated paternal allele but do not show other imprint-ing GNAS defects. The affected individuals show a loss of imprinting in the entire maternal allele. The presence of similarly large deletions at the NESP55 DMR has been excluded in a number of sporadic PHP 1b cases.13,133 However, a different 4.2-kb deletion has been identified in the affected individuals of a different PHP 1b kindred who displayed broad GNAS imprinting defects (see Fig. 66-8).205 This new deletion also includes antisense exons 3 and 4 but spares exon NESP55, overlapping with the previously identified two deletions by about 1.5 kb.205 Thus, these identified deletions reveal the putative loca-tion of another control element required for the imprint-ing of the entire maternal GNAS allele.

A more recent study revealed a 19-kb genomic deletion removing exon NESP55 and a large portion of the intron between NESP55 and antisense exon 4 in a kindred with AD-PHP 1b with isolated loss of A/B methylation.206 Since these patients lacked broad GNAS methylation abnormalities, the identified deletion points to the exis-tence of another control element of A/B methylation, without effects on other DMRs. The study of a mouse model in which Nesp55 transcription was prematurely truncated revealed loss of imprinting at exon 1A (the equivalent of human exon A/B) and, less consistently, the antisense promoter and exon XL.207 Taken together with the genetic findings in PHP 1b patients, it appears that the establishment of methylation imprints on the maternal GNAS allele is essential for allowing expression of Gsα in the proximal renal tubule and other tissues, including the thyroid gland, in which this GNAS product is derived only from the maternal allele. Furthermore, the evidence that exon A/B methylation requires transcription from the NESP55 promoter207 raises the possibility that even small deletions or even point mutations can prevent the genera-tion of NESP55 pre-mRNA, thus leading to PHP 1b.

Sporadic disease appears to be the most frequent cause of PHP 1b. These cases all show broad GNAS methyla-tion defects. However, the maternal allele is frequently shared between affected and unaffected siblings, suggest-ing that these cases may carry small de novo mutations in this region. However, in some families the affected female passed either allele #1 or allele #2 to their children, who are all unaffected by PHP 1b and show no methylation abnormality at the GNAS locus.208,209 It is therefore possi-ble that some of the sporadic PHP 1b cases carry homozy-gous or compound heterozygous mutations in an entirely different genomic location resulting in a putative auto-somal recessive mode of inheritance.208 Consistent with this conclusion, few patients affected by imprinting disor-ders without obvious PTH-resistance show GNAS meth-ylation changes.210,211 Moreover, methylation changes

in some other imprinted genomic loci have been revealed in a study of sporadic PHP 1b cases.212 It has also been suggested that the broad GNAS imprinting defects observed in sporadic PHP 1b patients result from stochas-tic defects in the regulation of imprinting.201

Despite having distinct epigenetic abnormalities at the GNAS locus (i.e., isolated A/B loss of imprinting versus broad imprinting defects that involve exon A/B and at least one other GNAS DMR), PHP 1b patients seem to have similar clinical findings with respect to serum cal-cium, phosphate, and PTH levels.13 Analysis of 20 fami-lies in which the affected individuals show an isolated loss of A/B imprinting reveals that a significant portion of such familial cases are asymptomatic at the time of diagnosis. In some of these cases, the diagnosis was made only based on elevated serum PTH. Comparison of male and female patients among sporadic PHP 1b cases who exhibit GNAS imprinting defects at two or more GNAS DMRs also reveals that female patients have significantly higher serum PTH levels than male patients, suggesting that hormone resistance is more severe in females.13

By definition, PHP 1b patients do not show AHO fea-tures. However, some recent reports identified patients who carry genetic and epigenetic defects associated with PHP 1b yet present with mild AHO features, particularly the shortness of metacarpal bones.27,202,213-215 This may suggest that Gsα imprinting occurs in more tissues than currently recognized, though there are alternative expla-nations. Considering that individual AHO features can be observed in other disorders, the presence of AHO fea-tures may be unrelated to the molecular genetic defects underlying PHP 1b in these cases. In one case, the mother of two affected siblings with short metacarpals and loss of A/B methylation also exhibited short metacarpals despite lacking any GNAS epigenetic abnormalities,202 suggesting that the finding of short metacarpals is unre-lated to the epigenetic defect in that family. In addition, the observed coexistence of GNAS imprinting defects and AHO can result from a large genomic deletion compris-ing at least the promoter of Gsα and one or more differ-entially methylated regions. In fact, such a large deletion leading to misdiagnosis of PHP 1b has been discovered in a case with apparent loss of A/B methylation and AHO features.216

PSEUDOHYPOPARATHYROIDISM TYPE 2Dissociation regarding the impairment of PTH-induced nephrogenous cAMP formation and phosphaturia (i.e., PHP 2) appears to be the least common form of PHP. Although typically sporadic, a case with familial form of PHP 2 type has been reported,217 and several reports describe evidence for a self-limited form of this disease in newborns, which could indicate that it is transient in nature.35,218-220 The molecular defect and pathophysi-ologic mechanisms underlying this PHP variant remain to be discovered. Because the defect underlying PHP 2 is associated with normal cAMP generation in response to exogenous PTH, it was postulated that it is caused by molecular defects that involve downstream of cAMP generation, such as protein kinase A.19 In fact, mutations


in the regulatory subunit of protein kinase A have been identified in some patients who show, in association with characteristic skeletal abnormalities, biochemical defects similar to those seen in PHP 2 (see later). Alternatively, the PTH signaling pathways that utilize other G proteins, such as Gq or G11, may be defective in patients with PHP 2. The signaling mediated by the Gq/G11 pathway involves activation of phospholipase C, which in turn leads to the formation of second messengers inositol 1,4,5 tris-phosphate (IP3) and diacylglycerol (DAG). This sig-naling pathway, which results in the stimulation of PKC and an increase in intracellular calcium ions, was shown to be important in sustaining the phosphaturic actions on PTH, as recently shown for mice expressing a PTHR-1 mutant that fails to activate IP3/PKC signaling.221,222 Serum calcium levels, which may affect the efficient utili-zation of intracellular calcium signaling pathways, appear to be important for restoring PTH responsiveness in PHP 2, as shown in some patients who normalized their phosphaturic response to PTH following normalization of serum calcium.223 It is also possible that the sodium-phosphate transporters in the proximal renal tubule are nonresponsive to PTH, thereby resulting in a defective phosphaturic, but not cAMP, response to exogenous PTH. Such a defect, however, should preserve the action of PTH on 25(OH)D-1-α-hydroxylase and lead to normal serum 1,25(OH)2D, unless it is combined with vitamin D deficiency. Hypocalcemia as a result of vitamin D defi-ciency has also been associated with PTH resistance that entailed the phosphaturic effect of this hormone without altering its potential to raise urinary cAMP,224 suggest-ing that some PHP 2 cases may in fact reflect vitamin D deficiency.225,226

ACRODYSOSTOSIS WITH HORMONAL RESISTANCEA recent report has described three patients who had resistance to PTH and some other hormones but showed unimpaired urinary cAMP excretion in response to exog-enously administered recombinant PTH.227 Consistent with resistance to the actions of PTH downstream of cAMP/PKA, PTH-induced urinary phosphate excretion was lost, and PTH-induced inhibition of urinary fractional calcium excretion was impaired. These unrelated patients, who could thus be classified as having a variant of PHP 2 based on their renal PTH responsiveness, also displayed acrodysostosis, a skeletal dysplasia that resembles, but is more severe than, the skeletal defects observed in AHO. Acrodysostosis, also known as Maroteaux-Malamut syn-drome, includes severe bradydactyly, peripheral dysosto-sis, and nasal hypoplasia.228-230 This disorder is termed acrodysostosis with hormonal resistance (ADOHR). Analysis of DNA from these patients revealed a recur-rent heterozygous nonsense mutation of the gene encod-ing cAMP-dependent protein kinase (PKA) type 1 alpha regulatory subunit (PRKAR1A; p.Arg368X). This muta-tion leads to a mutant PRKAR1A protein missing the last 14 C-terminal amino acids. Accordingly, cAMP cannot induce dissociation of this mutant regulatory subunit from the catalytic subunit, thereby keeping PKA in its inactive form. Several other patients with ADOHR have

been described in more recent studies in which additional PRKAR1A mutations have been documented.44,45,231-233 Note that the distal tubular actions of PTH appear to be impaired in patients with ADOHR, unlike in patients with PHP 1a and PHP 1b, consistent with the normal biallelic expression of Gsα in the distal renal tubule. Neverthe-less, the similarity between this disorder and PHP 1a with respect to the repertoire of hormone resistance is quite striking and highlights the critical nature of the cAMP signaling pathway in the actions of these hormones (Fig. 66-9).234

Genetic defects in another protein within the cAMP signaling pathway, cAMP phosphodiesterase type 4D (PDE4D), have also been identified upon exome sequenc-ing of DNA from some patients with acrodysostosis.44,45 These heterozygous missense mutations in PDE4D are associated with acrodysostosis, but the patients usually lack endocrine abnormalities. Other missense mutations of PDE4D have been identified in additional patients who also show acrodysostosis in the absence of hormone resis-tance (termed acrodysostosis without hormonal resis-tance [ADOP4]).232,235 Thus, PDE4D mutations are not associated with renal PTH resistance, and accordingly, unlike patients with PRKAR1A mutations, who demon-strate elevated basal urinary cAMP levels, patients with PDE4D mutations show normal basal and PTH-induced urinary cAMP level.232 The biochemical mechanisms underlying ADOP4 have yet to be elucidated. It is pre-dicted, however, that the PDE4D mutations are gain-of-function, given that PDE4D limits the intracellular level of cAMP.236

TREATMENTThe primary goal of treatment entails correction of abnormal serum biochemistries that result from PTH and, in some cases, other hormone resistance, such as TSH resistance leading to hypothyroidism, which can be treated by thyroid hormone replacement. GH deficiency can also be treated with recombinant human GH (rhGH) and is found to be efficacious in prepubertal patients with PHP 1a.237 PPHP patients who carry de novo mutations on the paternal GNAS allele are often diagnosed late in life (often when the children of female PPHP are diag-nosed with PHP 1a), making it difficult to assess GH deficiency and thus the benefits of rhGH treatment in PPHP patients. Clinical management of hypocalcemia in patients with PHP is less difficult than in patients with hypoparathyroidism, because the distal tubular actions of PTH in PHP patients are not impaired, providing sufficient calcium reabsorption from the glomerular fil-trate. The treatment involves oral calcium supplements and activated vitamin D analogues, such as 1,25(OH)2D (calcitriol). Note that the active form of vitamin D is required because of the lowered capacity of the proximal tubule to convert 25(OH)D into the biologically active 1,25(OH)2D. In addition, treatment of patients with PTH resistance should aim at keeping the serum PTH level within or close to the normal range rather than sim-ply avoiding symptomatic hypocalcemia, since persistent elevation of serum PTH will increase bone resorption


and may eventually lead to hyperparathyroid bone dis-ease.238,239 Due to intact PTH actions in the distal tubule, urinary calcium levels are usually low, and affected individuals do not have a significant risk for developing kidney stones and nephrocalcinosis. In fact, during the course of treatment, elevation of urinary calcium typi-cally does not occur. Nevertheless, blood chemistries and urinary calcium excretion in patients undergoing treat-ment should be monitored annually, but more frequently during pubertal development and once skeletal growth is completed, as the requirements for treatment with cal-cium and 1,25(OH)2D may need to be reduced.

SUMMARYPHP refers to a group of disorders characterized by PTH resistance associated with hypocalcemia, hyperphospha-temia, and elevated serum PTH. Proximal tubular resis-tance to PTH is the most prominent hormonal defect but, depending on the underlying genetic defect, resistance to other hormones is also observed. PTH and these other hormones all exert their actions via receptors that couple to Gsα. The primary genetic cause of PHP 1 and related disorders is mutations that affect the complex GNAS locus, the gene encoding Gsα. These mutations result in decreased expression/activity of Gsα but also affect some

of the other gene products derived from GNAS. The nature and the parental origin of the GNAS mutation is an important determinant of the clinical manifestations. Mutations that affect coding Gsα exons lead to broader clinical abnormalities than mutations that disrupt GNAS imprinting. Due to the tissue-specific imprinting of Gsα, hormone resistance develops only after maternal inheri-tance. AHO typically occurs after both maternal and paternal inheritance of coding Gsα mutations, although the repertoire of some AHO features also follows an imprinted mode of inheritance. PPHP is used to describe those patients with AHO who lack hormone resistance. AHO features vary markedly. An extreme manifesta-tion of AHO is progressive osseous heteroplasia, which appears to develop when Gsα mutations are present on the paternal allele, possibly in combination with another mutation. PHP 2 is rare, and the molecular determinants of at least one variant with skeletal dysplasia involve the regulatory subunit of PKA. • For your free Expert Consult eBook with biblio-

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Acrodysostosis withhormonal resistance (ADOHR)

Inactive PKA

ATP

Blomstrand’sdisease

Adenylatecyclase

PHP 1a and PHP 1b:hormonal resistance ± AHO

cAMP

Regulatorysubunit

(PRKAR1A)Catalyticsubunit

AMP

ActivePKAcAMP

Cellularevents

Acrodysostosiswithout hormonal

resistance (ADOP4)

β/γ

Gsα

PDE4D

RR

R C

C

R C

C

Figure 66-9 Mutated components of the cAMP signaling pathway in PHP and acrodysostosis. Gsα is essential for stimulation of cAMP genera-tion, which binds to the regulatory subunits of PKA and thereby renders the catalytic subunits active. cAMP-specific phosphodiesterase 4D (PDE4D) hydrolyzes cAMP and thereby controls its intracellular levels. Homozygous or compound heterozygous mutations in the PTH/PTHrP receptor (PTHR-1) are the cause of Blomstrand’s lethal chondrodysplasia, while maternally inherited, heterozygous inactivating mutations in Gsα-coding GNAS exons or deletions disrupting the imprinting of GNAS lead to PHP 1a or PHP 1b, respectively, resulting in resistance to PTH and frequently other hormones. Heterozygous mutations in PRKAR1A also lead to resistance to multiple hormones and are the cause of acrodysostosis with hormonal resistance (ADOHR), while mutations in PDE4D result in acrodysostosis without hormonal resistance (ADOP4). (Modified from Silve C, Jüppner H. Genetic disorders caused by mutations in the PTH/PTHrP receptor and downstream effector molecules. In: Bilezikian JP (ed.), The parathyroids. Elsevier, 2015;587-605.)

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Chapter 66 – Pseudohypoparathyroidism, Albright’s ...66 PSEUDOHYPOPARATHYROIDISM, ALBRIGHT’S HEREDITARY OSTEODYSTROPHY, AND PROGRESSIE OSSEOUS 1149 clinical manifestations that