c h a p t e r 66 Pseudohypoparathyroidism, Albright’s Hereditary Osteodystrophy, and Progressive Osseous Heteroplasia: Disorders Caused by Inactivating GNAS Mutations*

Murat Bastepe and Harald Jüppner

CHAPTER OUTLINE

DIAGNOSIS AND PROGRESSION OF PTH PSEUDOHYPOPARATHYROIDISM TYPE 1b, 1155 RESISTANCE, 1148 PSEUDOHYPOPARATHYROIDISM TYPE 2, 1157 PSEUDOHYPOPARATHYROIDISM TYPE 1a, 1148 ACRODYSOSTOSIS WITH HORMONAL THE COMPLEX GNAS LOCUS, 1151 RESISTANCE, 1158 CLINICALLY DISTINCT, GENETICALLY RELATED PHP TREATMENT, 1158 1a VARIANTS, 1153 SUMMARY, 1159 Pseudopseudohypoparathyroidism, 1153 Progressive Osseous Heteroplasia, 1154

KEY POINTS

• 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.

In 1942, Albright and colleagues1 described a group hyperphosphatemia in these patients resulted from of patients who displayed certain physical features, target-organ resistance to, rather than deficiency of, including obesity, short stature, brachydactyly, and PTH. Consistent with resistance to the actions of this cognitive impairment, combined with hypocalcemia hormone, it was later shown that patients affected by and hyperphosphatemia. In these patients, exoge- PHP have elevated concentrations of immunoreac- nous, biologically active parathyroid hormone (PTH) tive PTH.2 Subsequently, it was also shown that some extracts failed to result in a full phosphaturic response; patients affected by PHP have resistance toward other hence, the term pseudohypoparathyroidism (PHP) hormones; however, PTH resistance is the most promi- was introduced, indicating that hypocalcemia and nent feature of the disease.

*Chapter titles shaded in green indicate chapters dedicated predominantly to pediatric endocrinology content. 1147 1148 PART 6 PARATHYROID GLAND, CALCIOTROPIC HORMONES, AND BONE METABOLISM

The primary site of PTH resistance in PHP is the renal 1000 proximal tubule, as the actions of PTH in bone and the distal tubule appear normal.3-5 Patients with PHP have 300 reduced serum concentrations of 1,25-dihydroxyvitamin D [1,25(OH)2D], which is the main cause of hypocal- 6-9 cemia. Both low serum 1,25(OH)2D and hyperphos- 100 phatemia are the direct results of PTH resistance at the proximal tubule. Hyperphosphatemia is typically wors- 30 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 Urinary cAMP 10

PTH can prevent symptomatic hypocalcemia in some (nanomoles/100 mL GF) PHP patients due largely to its unimpaired actions on the 3 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 1 patients manifest hypocalcemia and, therefore, present 03060 120 180 with associated clinical findings. Minutes Figure 66-1 Time-course of urinary cAMP levels in response to PTH infusion. Upon injection of PTH, normal subjects (blue triangles) ex- DIAGNOSIS AND PROGRESSION OF PTH RESISTANCE 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 PTH exerts its actions by binding to a seven-transmem- response. (Adapted from Levine MA, Jap TS, Mauseth RS, et al. Activ- brane, G protein–coupled receptor (the PTH/PTH-related ity of the stimulatory guanine nucleotide-binding protein is reduced in protein receptor, PTHR1)15. Although the PTHR1 can erythrocytes from patients with pseudohypoparathyroidism and pseudop- couple to several different G proteins,16 most PTH actions seudohypoparathyroidism: Biochemical, endocrine, and genetic analysis are mediated primarily through the stimulatory G protein, of Albright’s hereditary osteodystrophy in six kindreds. J Clin Endocrinol Metab 62:497-502, 1986). which acts on adenylyl cyclases, thereby increasing the for- mation of intracellular second-messenger cyclic adenosine monophosphate (cAMP).15,17 PTH-induced cAMP forma- genetic testing was performed.25 Some patients present- tion is used as an important indicator of renal tubular PTH ing with seizures demonstrate epileptiform EEG changes function, since most PHP patients display an inadequate or and, because this activity typically responds to antiepilep- absent increase of urinary cAMP in response to exogenous, tic treatment, the diagnosis of PHP can be delayed.26,27 biologically active PTH (Fig. 66-1).18 In fact, the nephrog- As another complication of the changes in serum calcium enous cAMP response to the exogenously administered and phosphorous, brain imaging studies frequently show PTH is utilized as a test for establishing the diagnosis of intracranial calcifications in PHP patients.20,28-34 PHP, which is more sensitive than measuring the increase PHP is a congenital disorder, but only few reports in urinary phosphate excretion used in the original Ells- describe findings consistent with PTH resistance during worth-Howard test. However, currently used high-sensi- the neonatal period.35,36 Clinical manifestation of hypo- tivity PTH assays often suffice to make the diagnosis when calcemia typically occurs only later in childhood, suggest- serum PTH is elevated in the presence of hypocalcemia and ing that PTH resistance and the resultant changes in serum hyperphosphatemia. Nonetheless, depending on the nature calcium and phosphorous develop only gradually.37-40 In a of the nephrogenous response to exogenous PTH, PHP is longitudinal study of a child with PHP 1a (see later), cAMP subdivided into two main types. PHP type 1 is defined by response to PTH was found to be normal at 3 months of blunted urinary excretion of both cAMP and phosphate, age, whereas it was blunted at 2.6 years of age.38 In another and PHP type 2 is defined by blunted urinary excretion of PHP 1a case, a gradual decline of serum calcium, preceded phosphate only.1,18,19 by increasing serum phosphorous and PTH levels, was Signs and symptoms of decreased serum calcium level demonstrated.37 In addition, a PHP 1b patient diagnosed often reflect secondary prolongation of the QT interval by genetic analysis at birth (see later) was shown to have on EKG and increased neuromuscular excitability lead- normal serum PTH levels until 18 months of age, when ing to Trousseau’s and Chvostek’s signs as well as bron- an elevation of PTH was first detected despite normal chospasm. Although the most common manifestations serum calcium and phosphorous.39 It thus appears that of hypocalcemia include muscle tetany and spasms, find- PTH responses are intact during the early postnatal period ings vary markedly among patients. In more severe cases, despite the existence of the molecular defect underlying patients present with seizures. Other neurologic symp- PHP. The mechanisms that allow normal PTH signaling toms can also arise from hypocalcemia, and some patients during this developmental stage remain unknown. with PHP have initially been diagnosed with movement 20-24 disorders. In one report, two sisters with PHP type PSEUDOHYPOPARATHYROIDISM TYPE 1a 1b (see later) presented with paroxysmal kinesigenic choreoathetosis, and the diagnosis of PTH resistance in Of the two main PHP types, PHP type 1 is much more one sister was made after approximately 4 years of oral common. Various clinical variants of PHP type 1 have antiepileptic treatment when biochemical evaluation and been described based on the presence or absence of 66 PSEUDOHYPOPARATHYROIDISM, ALBRIGHT’S HEREDITARY OSTEODYSTROPHY, AND PROGRESSIVE OSSEOUS 1149

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

Additional Typical PTH Hormone AHO Resistance Resistance Features Genetic Defect PHP Yes Yes Yes Coding GNAS 1a/1c mutations PPHP No No Yes Coding GNAS mutations POH* No No No Coding GNAS mutations PHP 1b Yes Some No† Microdeletions cases affecting GNAS imprinting , patUPD20q, or unknown defects. PHP 2 Yes No No Unknown ADOHR Yes Yes Yes Coding PRKAR1A mutations ADOHR, Acrodysostosis with hormonal resistance; AHO, Albright’s hereditary osteodystrophy; PHP, pseudohypoparathyroidism; POH, A 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. 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 B C stature, brachydactyly, ectopic ossification, and men- tal impairment (Fig. 66-2). These features are termed Figure 66-2 Albright’s hereditary osteodystrophy (AHO). A, Short Albright’s hereditary osteodystrophy (AHO) and occur stature and obesity are among the typical features of AHO. B, Hand primarily in PHP patients who are now classified as hav- 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- ing pseudohypoparathyroidism type 1a (PHP 1a). The graph at 8 10/12 years of age. (A, Adapted from Albright F, Burnett brachydactyly observed in patients with AHO typically CH, Smith PH, Parson W. Pseudohypoparathyroidism—an example of involves the metacarpal and/or metatarsal bones and, “Seabright-Bantam syndrome.” Endocrinology 30:922-932, 1942.) 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 Furthermore, the severity of each feature differs vastly to shortened metacarpals, dimpling over the knuckles among the patients. In addition, individual AHO fea- of a clenched fist (Archibald sign) is often observed.42 tures are not unique to PHP, as they can be observed The shortening of the distal phalanx of the thumb, in other unrelated disorders, for example, acrodysos- however, is the most common skeletal abnormality tosis caused by PDE4D mutations44,45 or brachydac- (called murderer’s thumb or potter’s thumb), and some tyly–mental retardation syndrome caused by HDAC4 patients have shortening of all digits.43 Mental impair- mutations.46 The variable expressivity and the lack of ment is mild, often presenting as cognitive defects. It specificity of individual features can make the AHO is possible that the cause of mental impairment is the diagnosis challenging. While the coexistence of hor- deficiency of Gsα signaling in the brain. While hypocal- mone resistance in PHP 1a patients is often helpful, it cemia and/or hypothyroidism may also play a role in can also be misleading. This is particularly important this phenotype, correction of these biochemical defects for the differential diagnosis of different PHP forms does not prevent mental impairment in all cases. There characterized by the presence of AHO features alone is remarkable patient-to-patient variability in AHO, or hormone resistance alone (see later). even among patients that carry the same genetic muta- In addition to having PTH resistance and AHO, tion and belong to the same family (see later for a dis- patients with PHP 1a show clinical evidence that is consis- cussion of the underlying genetic defect). Some patients tent with target-organ resistance to other hormones. The may exhibit a single AHO feature only, such as obesity, most common additional hormone resistance involves the while others may present with multiple AHO features. actions of TSH, leading to hypothyroidism.47,48 In fact, 1150 PART 6 PARATHYROID GLAND, CALCIOTROPIC HORMONES, AND BONE METABOLISM unlike PTH resistance, which typically develops later in that disrupt pre-mRNA splicing (an extensive list of these life, TSH resistance can be present at birth.49-52 Resis- mutations can be found online at OMIM entry #139320 tance to gonadotropins and growth hormone–releasing at http://www.ncbi.nlm.nih.gov). Constitutional dele- factor has been reported,53-55 whereas resistance to other tions of the chromosomal arm containing GNAS have peptide hormones that also mediate their actions through also been identified.66 Consistent with this mutational Gsα-coupled receptors, such as vasopressin or ACTH, spectrum, Gsα level/activity is reduced by approximately does not appear to become clinically overt.54,56-60 50% in easily accessible tissues from PHP 1a patients, The genetic mutation that causes PHP 1a is located such as erythrocytes, skin fibroblasts, and platelets.48,67-80 within the Gsα-coding GNAS exons.61,62 A protein that is Deficiency of Gsα has also been demonstrated in renal essential for the actions of many hormones, Gsα primar- membranes from a patient with PHP.81 A complementa- ily mediates agonist-induced generation of intracellular tion assay is typically used for examining Gsα activity, cAMP. Activation of a stimulatory G protein–coupled involving patient-derived erythrocyte membranes and receptor by its agonist, such as PTH, leads to a GDP- membranes from turkey erythrocyte that lack endogenous GTP exchange on Gsα, causing dissociation of the latter Gsα (Fig. 66-4). Gsα hypofunction can also be detected in from Gβγ subunits (Fig. 66-3). This allows both Gsα and patient-derived platelets by using a functional test that Gβγ to stimulate their respective effectors. In its GTP- examines the ability of prostaglandin E1 or prostacyclin bound state, Gsα can directly activate several different to inhibit platelet aggregation.82 The detection of reduced effectors, such as Src tyrosine kinase,63 and certain Ca- Gsα activity is important for the establishment of PHP 1a channels.64,65 Apart from these effectors, however, ade- diagnosis, particularly for cases in which genetic analysis nylyl cyclase is by far the most ubiquitous and the most fails to reveal a GNAS mutation. Reduction of Gsα activ- extensively investigated effector molecule stimulated by ity in PHP 1a is consistent with the fact that PTH and the Gsα. An integral membrane protein, adenylyl cyclase cat- other hormones with impaired actions in this disorder act alyzes the synthesis of the ubiquitous cAMP, which then via cAMP-mediated signaling pathways. triggers various cell-specific responses. The activation of Among the many different inactivating GNAS muta- adenylyl cyclase and other effectors by Gsα is regulated tions, a 4-bp deletion in exon 7 has been identified in by the intrinsic GTP hydrolase (GTPase) activity of Gsα. numerous families, representing a genetic “hot-spot.” Conversion of GTP into GDP results in the re-assembly of In addition, two different mutations are associated with the G protein heterotrimer and, thereby, prevents further additional phenotypes. A missense mutation in exon 13 effector stimulation (see Fig. 66-3). (A366S) was identified in two unrelated boys, who pre- Mutations identified in PHP 1a patients are heterozy- sented with PHP 1a and precocious puberty.83,84 This gous and scattered throughout all of the 13 GNAS exons mutant Gsα protein is temperature-sensitive and thus rap- encoding Gsα and the intervening sequences, including idly degraded at normal body temperature. The amino acid missense and nonsense amino acid changes, insertions/ substitution, however, renders the protein constitutively deletions that cause frameshift, and nucleotide alterations 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, α βγ which entails a repeated AVDT sequence at residues 366 to 369, generates an unstable but constitutively active Gsα GDP A

R 100 α α βγ 80 GDP GTP GDP 60

α + βγ 40 (% of control pool) GTP 20 Erythrocyte Gs α activity Figure 66-3 Heterotrimeric G protein activation cycle. The heterotri- meric complex is assembled at the basal state, with the α-subunit being 0 associated with a GDP molecule. Upon binding of an agonist (A) to its PHP 1a pseudoPHP PHP 1b 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 Figure 66-4 Gsα erythrocyte activity in PHP 1a, PPHP, and PHP guanine nucleotide exchange factor for the α-subunit. The GTP-bound 1b patients. The measurement is conducted by complementation of form of the α-subunit dissociates from the βγ subunits and thereby patient-derived erythrocyte membranes with membranes from S49cyc–­ stimulates its downstream effectors, which, in the case of Gsα, includes lymphoma cells, which genetically lack Gsα but retain all other com- adenylyl cyclase. Note that the free Gβγ dimer can also stimulate differ- ponents necessary for hormone-activated adenylyl cyclase response. ent downstream effectors. The intrinsic GTP hydrolase activity of the (Adapted from Levine MA. Hypoparathyroidism and pseudohypopara- α-subunit converts the GTP into GDP, resulting in the re-association thyroidism. In De Groot LJ, Jameson J, eds. Endocrinology, 4th ed. of the heterotrimer and, therefore, termination of effector stimulation. Philadelphia: Saunders, 2000, pp. 1133-1153.). 66 PSEUDOHYPOPARATHYROIDISM, ALBRIGHT’S HEREDITARY OSTEODYSTROPHY, AND PROGRESSIVE OSSEOUS 1151 mutant due to enhanced GDP-GTP exchange and reduced allows basal cAMP formation but is unable to trigger an GTPase activity. While hormone resistance results from increase in response to hormones. Hence, at least some the instability of the Gsα-AVDT mutant, the diarrhea is PHP 1c cases are allelic variants of PHP 1a. Although attributed to increased plasma membrane localization it remains possible that some patients who match the of the mutant protein in the small intestine epithelium. description of PHP 1c develop hormone resistance and Another pediatric case has been described in whom a de AHO due to a novel gene mutation that affects cAMP novo missense mutation (R231C) on the paternal allele production without functionally impairing Gsα itself was found together with a maternally inherited combina- (e.g., inactivating mutations affecting one of the adeny- tion of three C-to-T substitutions, resulting in aberrant lyl cyclases or activating mutations in the phosphodies- GNAS splicing.86 The patient with these compound het- terase), it appears unlikely that such putative mutations erozygous mutations had morbid obesity, TSH and PTH affect only few G protein–coupled receptors and thus resistance, and a prothrombotic state due to marked Gsα cause limited hormonal resistance as observed in PHP hypofunction in platelets. 1a and PHP 1c, but instead dictate the phenotype by the PHP 1c has been described as a distinct variant of PHP tissue-specificity of their expression levels. 1a,70, but the clinical and laboratory features of patients with PHP 1c are identical to those with PHP 1a, as they THE COMPLEX GNAS LOCUS have both AHO and multihormone resistance. In contrast to PHP 1a, however, biochemical assays demonstrate no GNAS is a complex locus giving rise to multiple different reduction in Gsα activity in erythrocytes obtained from coding and noncoding transcripts that show monoallelic, PHP 1c patients, suggesting the absence of mutations parent-of-origin–specific expression profilesFig. ( 66-5). within the Gsα gene. Nevertheless, recent molecular GNAS maps to the telomeric end of the long arm of chro- characterizations have revealed Gsα mutations at least in mosome 20 (20q13.2-20q13.3).88-90 Gsα is encoded by some PHP 1c patients. However, the Gsα mutants show 13 exons,91 but due to alternative pre-mRNA splicing, only impaired coupling to different G protein–coupled the Gsα transcript has several variants. The long and the receptors, yet show normal Gsα activity in complementa- short Gsα variants (Gsα-L and Gsα-S, respectively) differ tion assays that use non-hydrolyzable GTP analogues for from each other by the inclusion or exclusion of exon stimulation of Gsα activity rather than a receptor ago- 3;91-93 these Gsα variants are typically detected as 52- and nist.43,87 Thus, the mutant Gsα protein causing PHP 1c 45-kD protein bands on Western blots. Showing further

CH3 –– – Paternal

5 NESP5543 21 XL A20 A21 A/B 123N1456713

– CH3 CH3 CH3 Maternal

* Biallelic exons 2–13 (most tissues) exons 1

* exons 2–13 A/B

* Paternal exons 2–13 XL

AS exons 1–5 AS * Maternal exons 2–13 NESP55 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). 1152 PART 6 PARATHYROID GLAND, CALCIOTROPIC HORMONES, AND BONE METABOLISM complexity, each Gsα form either includes or excludes a the XLαs transcript is included in the translated product, CAG trinucleotide (encoding serine) at the start of exon resulting in a protein that is partially identical to Gsα.111 4. Small, but potentially important, differences have been Consistent with its structural similarity to Gsα, XLαs can reported between the activities of Gsα-L and Gsα-S. For mediate receptor-activated adenylyl cyclase stimulation example, Gsα-L has been shown to display greater abil- in transfected cells and in transgenic mice.43,115,121,122 In ity to mediate receptor signaling than Gsα-S when par- fact, it appears to be basally more potent than Gsα when tially purified proteins from rabbit liver were examined,94 expressed at levels comparable to the latter and is able to although the opposite finding was reported upon the use prolong PTH signaling in transfected cells.120,123 How- of cultured pancreatic islet cells.95 Furthermore, the GDP ever, the phenotype of mice with targeted ablation of XLαs release rate from Gsα-L appears to be approximately suggests that XLαs has unique, albeit as-yet-undefined, twofold higher than Gsα-S,96 and, accordingly, fusion cellular functions. These mice show high early postnatal proteins involving the β2-adrenergic receptor and Gsα-L mortality due to poor adaptation to feeding and impair- have shown higher constitutive activity than those involv- ment in the glucose and energy metabolism,118 in contrast ing the receptor and Gsα-S.97 Moreover, differences in to Gsα knockout mice, which to a large extent recapitu- the subcellular targeting of these two Gsα variants have late the findings in patients with PHP 1a.124,125 Findings been reported.98-100 Currently, it remains unclear whether from adult mice with disrupted XLαs expression indicate these differences translate into biologically significant that this protein is a negative regulator of increased sym- effects, such as divergence in the variety of effectors and/ pathetic nervous system activity in mice.126,127 or the efficiency of effector activation. Nonetheless, a The paternal GNAS also gives rise to two additional single nucleotide insertion in exon 3 leading to frameshift transcripts. From the sense strand originates the A/B tran- and early termination has been recently identified in two script (also termed 1A or 1′), which, similar to NESP55 siblings affected by a mild form of PHP 1a.101 The mild- and XLαs, utilizes an upstream promoter and an alter- ness of the phenotype is consistent with generation of native first exon (exon A/B) that splices onto Gsα exons one of the two main Gsα variants (i.e., Gsα-S). It remains 2 to 13.128,129 Exon A/B does not comprise an in-frame unknown whether this exon 3 mutation impairs agonist translation initiation codon but, as demonstrated in vitro, responses in an effector- and tissue-specific manner, and the translation can be started through the use of an AUG this possibility depends on the putative effector selectivity located within exon 2, thereby giving rise to an N-termi- and relative expression levels of Gsα-L and Gsα-S in dif- nally truncated protein that localizes to the plasma mem- ferent tissues. brane when transiently expressed in cell lines.129 Until Recent studies have revealed that GNAS gives rise to recently, no evidence has supported the existence of an multiple additional gene products that show parent-of- endogenous A/B protein, and therefore, it was thought origin–specific, monoallelic expression. Besides Gsα, at that the A/B transcript was a noncoding RNA. Endo­ least two translated GNAS transcripts exist, using distinct genous A/B protein has recently been detected, however, upstream promoters and alternative first exons that splice in human fetal kidney by Western blot using an antibody onto exons 2 to 13 encoding Gsα. The most upstream against the C-terminal end of Gsα.130 It remains possible promoter relative to the Gsα promoter drives expression that A/B has functions both as a protein and as a tran- of a neuroendocrine secretory protein with an apparent script regulating the expression within the GNAS locus molecular mass of 55,000 (NESP55).102 The paternal (see later). Another paternal GNAS transcript is derived NESP55 promoter is methylated, and the transcription from the antisense strand.131,132 The GNAS antisense occurs from the maternal allele.103,104 In humans, NESP55 transcript (GNAS-AS1), which is formed in humans by protein is encoded by a single exon, so that Gsα exons 2 at least six primary exons that show multiple alternative to 13 compose the 3′ untranslated region.103,104 Expressed splicing patterns,131,133 is also considered to be noncod- in neuroendocrine tissues, peripheral and central nervous ing. Data from mouse models indicate that GNAS-AS1 system, and some endocrine tissues,102,105-108 NESP55 plays a critical role in silencing the paternal NESP55 pro- is a chromogranin-like protein associated with the con- moter in cis.134 The promoter of GNAS antisense tran- stitutive secretory pathway.109 Loss of the expression of script is immediately upstream of the promoter of XLαs. NESP55 protein does not seem to have an overt clinical Although the promoters of XLαs and antisense tran- outcome in humans, as evidenced from patients with script are located together within a large differentially PHP type 1b (see later). However, its disruption in mice methylated region (DMR), the female germ line–specific result in a subtle behavioral phenotype characterized by imprint is established at the antisense promoter only.135 increased reactivity to novel environments.110 Transcrip- The A/B promoter is likewise methylated in the female tion from the NESP55 promoter is likely to play a role in germ line, but not in the male germ line.136 Thus, the two the regulation of GNAS imprinting (see later). germ line imprint marks at the GNAS locus include the Another GNAS product is XLαs, which is expressed promoters of the antisense and A/B transcripts. Accord- from the paternal allele.104,111,112 XLαs is expressed abun- ingly, data from different genetically manipulated mouse dantly in various parts of the brain and neuroendocrine models show that these noncoding transcripts are essen- tissues, and its expression can be readily detected in a tial for the regulation of imprinted gene expression variety of other fetal and postnatal tissues.111,113-120 XLαs from GNAS.134,137,138 Imprinted A/B transcription (i.e., also uses a distinct upstream promoter and a unique first expression from only the non-methylated maternal allele) exon that splices onto Gsα exons 2 to 13.104,112 Unlike is particularly important for the development of hormone in the NESP55 transcript, however, the latter portion of resistance seen in patients with PHP 1b (see later). 66 PSEUDOHYPOPARATHYROIDISM, ALBRIGHT’S HEREDITARY OSTEODYSTROPHY, AND PROGRESSIVE OSSEOUS 1153

Unlike the promoters of NESP55, antisense, XLαs, and p A/B transcripts, the promoter of Gsα is not differentially hormone resistance and methylated and, accordingly, Gsα expression is biallelic m AHO, i.e., PHP 1a 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- p nisms, so that the maternal allele is the predominant m AHO only, i.e., PPHP source of Gsα expression. These tissues include the renal proximal tubule, thyroid, pituitary, and gonads.125,142-144 Figure 66-6 The effect of paternal Gsα silencing in the development of hormone resistance. Gs is biallelic in most tissues, and a heterozy- Although devoid of differential methylation, the Gs pro- α α gous inactivating Gsα mutation therefore causes an approximately 50% moter exhibits parent-of-origin–specific histone modifica- reduction in Gsα activity/expression regardless of the parent of origin tions in those tissues where it is monoallelic. The active of the mutation. However, in some tissues, such as the proximal tubule maternal Gsα promoter shows a greater ratio of tri- to and thyroid gland, paternally inherited Gsα transcript is silenced (X). 4 Thus, if a Gsα mutation (black square) is inherited from a female indi- di-methylated histone-3 Lys compared to the silenced vidual, this mutation nearly abolishes the expression or the activity of paternal promoter in the proximal tubule, whereas the Gsα in those tissues, thus leading to hormone resistance (PHP 1a). In amount of methylated histones is similar in maternal and contrast, upon paternal inheritance, the same mutation Gsα does not paternal Gsα promoters in liver, a tissue in which Gsα is lead to a significant change in the expression/activity, and thus, the hor- biallelic.145 As discussed later, the tissue-specific, paternal mone responses are unimpaired. Paternal and maternal Gsα alleles are depicted by white or grey rectangles. Gsα silencing has a key role in the development of PTH resistance in patients with PHP 1a and PHP 1b. resistance involves only a small subset of tissues despite CLINICALLY DISTINCT, GENETICALLY RELATED PHP the involvement of Gsα signaling in a multitude of physi- 1a VARIANTS ologic responses. Hormone resistance is observed in those tissues where Gsα is imprinted, such as the proximal renal Pseudopseudohypoparathyroidism tubule and the thyroid gland, while hormone responses Physical abnormalities similar to those observed in patients are unimpaired in those tissues where Gsα is biallelic, such with PHP 1a but without evidence for an abnormal as the distal renal tubules. The role of tissue-specific Gsα regulation of calcium and phosphate homeostasis were imprinting in the development of PTH resistance has been first reported in 1952.146 Because of the lack of an abnor- demonstrated through the generation of mice heterozy- mal regulation of mineral ion homeostasis, the name gous for maternal or paternal disruption of GNAS.142 A pseudopseudohypoparathyroidism (PPHP) was coined recent study furthermore showed that the silencing of the to describe this disorder.146 Interestingly, patients with paternal Gsα allele in the renal proximal tubule develops PPHP also carry GNAS mutations that lead to dimin- after the early postnatal period in mice, thus providing ished Gsα function, and these mutations can be found in a plausible explanation for the finding that the manifes- the same kindred as those with PHP 1a. However, both tation of PTH resistance occurs mostly after infancy in disorders are never seen in the same sibling kinship, and patients with PHP 1a and PHP 1b.151 a careful analysis of multiple families has revealed that The finding that most AHO features develop regardless the mode of inheritance of each disorder depends on the of the parent transmitting a Gsα mutation has led to the gender of the parent transmitting the Gsα mutations.147 hypothesis that the inheritance of AHO is due to Gsα hap- Thus, an inactivating Gsα mutation causes PHP 1a (i.e., loinsufficiency in various tissues, which appears to be true hormone resistance and AHO) after maternal inheritance, in certain settings. PTHrP-induced cAMP generation is whereas the same mutation on the paternal allele results critical for proper control of hypertrophic differentiation in PPHP (AHO only). Most AHO features, except obesity of growth plate chondrocytes,152 and Gsα haploinsuffi- and mental retardation, appear to develop regardless of ciency has been demonstrated in this tissue through the the parent of origin, and it is therefore primarily hormone study of mice chimeric for wild-type cells and mutant cells resistance that displays an imprinted mode of inheritance. heterozygous for disruption of GNAS exon 2.153 Regard- Recent studies have furthermore revealed that most PPHP less of the parental origin of the GNAS exon 2 disruption, patients are considerably smaller at birth, particularly if the mutant cells displayed premature hypertrophy com- their inactivating Gsα mutation is located in GNAS exons pared to their wild-type neighbors, although the paternal 2 to 13 of the paternal allele.148-150 disruption (i.e., loss of one Gsα allele combined with a The tissue-specific imprinting of Gsα expression can complete loss of XLαs) resulted in significantly more pre- explain the parent-of-origin–specific inheritance of hor- mature hypertrophy than the maternal disruption (loss of mone resistance. In those tissues where Gsα expression is one Gsα allele only). Thus, the brachydactyly and/or short paternally silenced (i.e., Gsα is expressed exclusively or stature observed in the context of AHO likely result from predominantly from the maternal allele), an inactivating diminished Gsα signaling in growth plate chondrocytes. mutation located on the paternal allele is not predicted to While these data correlate well with the notion that AHO alter Gsα function, whereas the same mutation located develops after both maternal and paternal inheritance of on the maternal allele is predicted to abolish Gsα func- a Gsα mutation, recent evidence suggests that individual tion completely (Fig. 66-6). The tissue-specific imprint- AHO features can also be subject to imprinting. A care- ing of Gsα expression also explains why the target organ ful analysis of multiple patients with PHP 1a and PPHP 1154 PART 6 PARATHYROID GLAND, CALCIOTROPIC HORMONES, AND BONE METABOLISM revealed that obesity is primarily a feature of PHP 1a extraskeletal ossifications that involve deep connective patients, developing after maternal inheritance.154 Con- tissue and skeletal muscle156,157 (Fig. 66-7). In POH, the sidering that Gsα is biallelic in the white adipose tissue,141 ectopic bone is primarily intramembranous, as opposed it was proposed that Gsα may also be imprinted (pre- to a similar disease termed fibrodysplasia ossificans pro- dominantly maternal expression) in areas of the central gressiva (FOP) in which extraskeletal bone formation nervous system that control satiety and body weight.154 occurs via endochondral mechanisms, and is accompa- A recent report has also demonstrated that cognitive nied by skeletal malformations.158,159 Few patients with impairment is more prevalent in PHP 1a than in PPHP, POH demonstrate AHO features and, consistent with thus indicating that tissue-specific Gsα imprinting may the occasional coexistence of these two sets of clinical involve additional brain regions.155 On the other hand, defects, heterozygous inactivating Gsα mutations have imprinted inheritance has not been reported regarding been identified as a cause of POH.160-162 Several of the short stature, despite the finding that Gsα is maternally identified mutations are identical to those found in PHP expressed in the pituitary gland143,144 and that PHP 1a 1a/PPHP kindreds.161,162 Gsα activity and downstream patients display GHRH resistance that results in growth signaling has been implicated in the control of osteo- hormone (GH) deficiency.54,55 Conversely, as outlined ear- genic differentiation. Patients who are mosaic for hetero- lier, the small for gestational age phenotype is associated zygous GNAS mutations that result in constitutive Gsα more strongly with Gsα mutations on the paternal allele activity develop fibrous dysplasia of bone characterized than with Gsα mutations on the maternal allele.148,150 by irregular woven bone disrupted by fibrous tissue.163 A single study also showed that PPHP patients carrying Moreover, in human mesenchymal stem cells, reduction mutations within exons 2 to 13 are born smaller than of Gsα protein levels has been shown to cause osteo- those carrying mutations within exon 1,150 thus impli- genic differentiation, while inhibiting the formation of cating both Gsα haploinsufficiency and the deficiency of adipocytes.164,165 In addition, Runx2, a key regulator a paternally expressed GNAS product, such as XLαs, in of osteoblast-specific gene expression, appears to sup- the pathogenesis. Future analyses of patients with PHP press Gsα expression.166 Thus, osteoprogenitor forma- 1a and PPHP will be helpful in determining the relative tion and the early stages of osteoblastic differentiation roles of genomic imprinting and haploinsufficiency in the require reduced levels of cAMP signaling, consistent development of individual AHO features. with the association of inactivating Gsα mutations with the severe ectopic bone formation observed in POH. Progressive Osseous Heteroplasia Because of the presence of GNAS mutations in both A disorder termed progressive osseous heteropla- AHO and POH, it appears that additional factors, such as sia (POH) has been described in patients with severe 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 B 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. A C N Engl J Med 346:99-106, 2002). 66 PSEUDOHYPOPARATHYROIDISM, ALBRIGHT’S HEREDITARY OSTEODYSTROPHY, AND PROGRESSIVE OSSEOUS 1155 factors may determine the penetrance and severity of the expression in the thyroid gland.144,177 Nevertheless, evi- ectopic ossifications in these patients that show approxi- dence for resistance to other hormones, such as gonado- mately 50% loss of Gsα activity. Nevertheless, clinical tropins, whose actions also involve tissues in which Gsα and genetic data demonstrate several important differ- is imprinted, has not been reported for PHP 1b patients. ences between patients with AHO and those with POH. A study assessed growth hormone response to GHRH First, the ectopic bone in AHO is limited to subcutane- plus arginine stimulation in PHP 1b, revealing a normal ous tissue. In addition, in nearly all patients with POH, response in 9 of 10 patients.178 On the other hand, in the severe ectopic bone formation is isolated (i.e., other addition to PTH and mild TSH resistance, hypourice- typical AHO features are not manifest).162,167 Moreover, mia due to increased fractional excretion of uric acid has mutations leading to isolated POH are inherited from been reported in the affected individuals of two unrelated male obligate gene carriers only (i.e., inheritance pattern PHP 1b kindreds.27,179 This finding implicates impaired is exclusively paternal).162 In fact, in one large kindred, PTH actions in the development of hypouricemia in these paternal inheritance of a GNAS mutation caused POH, patients, an interpretation that is consistent with two while maternal inheritance of the same mutation caused previous reports describing hyperuricemia in association typical AHO findings (without severe heterotopic ossifi- with hyperparathyroidism.180,181 However, hypouricemia cation).162 These findings indicate that the disease mecha- resolved in one of the PHP 1b kindreds following treat- nism underlying POH is significantly different from that ment with calcium and calcitriol.27 underlying AHO and that deficiency of a GNAS product Patients with PHP 1b display normal Gsα bioactivity/ with exclusive paternal expression, such as XLαs, contrib- levels in easily accessible tissues. Accordingly, coding Gsα utes to the molecular pathogenesis of POH. In addition, a mutations are ruled out in these patients. In one family, recent study analyzing 12 patients with POH has revealed however, a mutation located in exon 13 (in-frame deletion that the lesions follow a predominantly dermomyotomal of Ile,382 del382Ile) was reported, leading to the uncoup­ distribution that often shows a lateralization bias.168 ling of Gsα from the PTHR-1 but not other receptors that Based on these findings, which redefine the clinical defini- were expressed in LLCPK cells, including the TSHR, LHR, tion of this disorder, it was hypothesized that the patho- and β-adrenergic receptor,182 which are of renal origin and genesis involves progenitor cells of somitic origin, which express endogenous Gsα. These findings suggested an iso- may undergo loss of heterozygosity at the GNAS locus lated PTH resistance as seen in PHP 1b, leading to the con- and thereby cause severe or complete Gsα deficiency.168 clusion that the del382Ile mutation in Gsα represents a rare cause of PHP 1b. However, this conclusion has been ques- PSEUDOHYPOPARATHYROIDISM TYPE 1b tioned, as the use of transfected mouse embryonic fibroblasts null for endogenous Gsα showed that del382Ile leads to Another form of PHP was described by Peterman and uncoupling from not only PTHR-1 but also the β-adrenergic Garvey169 and by Reynolds and associates.170 Now receptor.43 Because of a lack of Gsα mutations and because known as pseudohypoparathyroidism type 1b (PHP Gsα activity/levels in easily accessible tissues are normal 1b), this PHP form is characterized by the presence of in PHP 1b patients, inactivating mutations that affect the PTH-resistant hypocalcemia and hyperphosphatemia, gene encoding PTHR-1 was considered in the past.183 How- but without evidence of AHO in most cases. In addition ever, several different studies have excluded such mutations to increased serum PTH, patients with PHP 1b can dem- as the cause of this disease.184-187 Instead, homozygous or onstrate elevated serum alkaline phosphatase activity, compound heterozygous, inactivating mutations of PTHR-1 which suggests normal PTH-dependent bone turnover.171 have been revealed as the cause of Blomstrand’s chondro- In fact, hyperparathyroid bone disease can be observed dysplasia, an embryonic lethal disorder with severe skeletal in association with PHP 1b, especially in patients with abnormalities.188 A homozygous PTHR-1 mutation was sporadic PHP 1b or in the index cases of the autosomal furthermore identified in patients with Eiken syndrome, an dominant form of PHP 1b (AD-PHP 1b). This occurs autosomal recessive skeletal dysplasia,189 and heterozygous less frequently, however, in PHP 1a since this variant is inactivating PTHR-1 mutations were observed in several associated with AHO features and is therefore diagnosed families with an autosomal dominant form of delayed tooth earlier in life. The intact PTH response in the bone is con- eruption190-193 (i.e., disorders without evidence for hypocal- sistent with the lack of Gsα imprinting in bone141 and led cemia and hyperphosphatemia because of impaired actions to the introduction of the term pseudohypo-hyperpara- of PTH). thyroidism (PHP-HPT).172-174 Based on genomewide linkage analysis, the genetic The hormone resistance observed in PHP 1b patients defect underlying PHP 1b maps to a region of chromo- develops only after maternal inheritance of the genetic some 20q13.3 that comprises the GNAS locus,175 but defect175 (i.e., the mode of inheritance is identical to the critical interval excludes all the coding GNAS exons, the hormone resistance in PHP 1a). PTH resistance and including those that encode Gsα.26,175 On the other hand, related changes in calcium and phosphate homeostasis patients with PHP 1b display epigenetic abnormalities are the major laboratory findings in PHP 1b, but some within the GNAS locus.26,194 The most consistent epi- PHP 1b patients also display mild hypothyroidism with genetic defect is a loss of imprinting at exon A/B (also slightly elevated TSH levels26,176-178 as well as some eleva- termed exon 1A), which is primarily found as an iso- tion in calcitonin level.176 Hypothyroidism, as in patients lated defect in familial PHP 1b cases.26,194 In addition, with PHP 1a, likely results from mild TSH resistance many sporadic and some familial PHP 1b cases show and is consistent with the predominantly maternal Gsα additional loss of imprinting at the DMR comprising the 1156 PART 6 PARATHYROID GLAND, CALCIOTROPIC HORMONES, AND BONE METABOLISM

XLαs and antisense promoters and a gain of imprint- kb.39 Recently, a maternally inherited 24.6-kb deletion ing at the DMR composing exon NESP55.13,194 These composing STX16 exons 2 to 8 has also been discov- abnormalities are associated with biallelic expression ered in another family with this disorder.203 Thus, the of A/B, XLαs, and antisense transcripts and silencing of disruption of STX16 appears to be the common genetic the NESP55 transcript. Together with the genetic link- defect in cases with isolated loss of exon A/B imprint- age data, these imprinting defects suggested that the ing. The parental origin of these STX16 deletions cor- mutation causing PHP 1b disrupts a regulatory element relates well with the mode of inheritance of PHP 1b. It that controls GNAS imprinting. However, evidence for is maternally inherited in affected individuals and pater- incomplete penetrance regarding the GNAS imprinting nally inherited in unaffected carriers. This gene encodes defects has been reported in one kindred, in whom some syntaxin-16, a member of the SNARE family proteins. individuals lacked loss of imprinting and were healthy However, STX16 does not appear to be imprinted,39,200 despite maternally inheriting the disease-associated hap- and it is therefore unlikely that the loss of one STX16 lotype.195 Thus, the imprinting abnormalities of GNAS copy leads to PHP 1b. Instead, since the maternal inheri- appear to be required for the development of PHP 1b. tance is associated with loss of exon A/B imprinting on Consistent with the importance of imprinting in the dis- the same allele, these deletions are presumed to disrupt ease mechanism, several patients with PHP 1b have been a cis-acting element controlling the establishment or reported to have paternal uniparental isodisomy involv- maintenance of exon A/B imprinting. Other than genetic ing whole or parts of chromosome 20 that comprise the evidence, however, no currently available data corrobo- GNAS locus.176,196-199 rate this prediction. A mouse model carrying a deletion In multiple familial PHP 1b cases with isolated exon equivalent to the 3-kb STX16 deletion in humans has A/B loss of imprinting, a unique 3-kb microdeletion been generated, but neither maternal nor paternal inher- at the centromeric neighboring STX16 locus has been itance of this genetic alteration causes PTH resistance identified200 (Fig. 66-8). The deleted region comprises or any alterations in GNAS imprinting204; animals with STX16 exons 4 to 6 and is flanked by two direct repeats, the homozygous Stx16 deletion are also healthy. It thus which may underlie the mechanism whereby this dele- appears plausible that the imprinting control element of tion occurs. This is consistent with the presence of the GNAS located within STX16 in the human is not pre- same microdeletion in many unrelated families with dif- cisely at the same location in the mouse. Nonetheless, ferent ethnic and racial origin.13,25,27,178,179,201,202 In a the absence of a phenotype in the Stx16 deletion mice single kindred, a different microdeletion within STX16 argues against a model in which syntaxin 16, the prod- has been reported, removing exons 2 to 4 and overlap- uct of this gene, is required in the oocyte for proper exon ping with the 3-kb microdeletion by approximately 1.3 A/B imprinting.

GNAS

CH3 – – – pat

STX16 NESP55

5 4A 4231XL A/B 1 2–13

– CH3 CH3 CH3 mat 4.2 kb 24.6 kb

4.0 kb/4.7 kb 3 kb

4.4 kb 19 kb

Figure 66-8 Mutations identified in AD-PHP 1b patients and their effects onGNAS 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 acis- 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 byhorizontal bars. 66 PSEUDOHYPOPARATHYROIDISM, ALBRIGHT’S HEREDITARY OSTEODYSTROPHY, AND PROGRESSIVE OSSEOUS 1157

In two unrelated familial cases of PHP 1b in whom in some other imprinted genomic loci have been revealed the affected individuals carried broad GNAS imprint- in a study of sporadic PHP 1b cases.212 It has also been ing defects, maternally inherited deletions of the entire suggested that the broad GNAS imprinting defects NESP55 DMR, including exons 3 and 4 of the antisense observed in sporadic PHP 1b patients result from stochas- transcript, have been identified (seeFig. 66-8).133 The tic defects in the regulation of imprinting.201 deletions are 4 kb and 4.7 kb large and have breakpoints Despite having distinct epigenetic abnormalities at the located in similar locations. The unaffected carriers in GNAS locus (i.e., isolated A/B loss of imprinting versus these families display an apparent loss of NESP55 methy­ broad imprinting defects that involve exon A/B and at lation due to the loss of this region from the normally least one other GNAS DMR), PHP 1b patients seem to methylated paternal allele but do not show other imprint- have similar clinical findings with respect to serum cal- ing GNAS defects. The affected individuals show a loss cium, phosphate, and PTH levels.13 Analysis of 20 fami- of imprinting in the entire maternal allele. The presence lies in which the affected individuals show an isolated of similarly large deletions at the NESP55 DMR has been loss of A/B imprinting reveals that a significant portion excluded in a number of sporadic PHP 1b cases.13,133 of such familial cases are asymptomatic at the time of However, a different 4.2-kb deletion has been identified diagnosis. In some of these cases, the diagnosis was made in the affected individuals of a different PHP 1b kindred only based on elevated serum PTH. Comparison of male who displayed broad GNAS imprinting defects (see Fig. and female patients among sporadic PHP 1b cases who 66-8).205 This new deletion also includes antisense exons exhibit GNAS imprinting defects at two or more GNAS 3 and 4 but spares exon NESP55, overlapping with the DMRs also reveals that female patients have significantly previously identified two deletions by about 1.5 kb.205 higher serum PTH levels than male patients, suggesting Thus, these identified deletions reveal the putative loca- that hormone resistance is more severe in females.13 tion of another control element required for the imprint- By definition, PHP 1b patients do not show AHO fea- ing of the entire maternal GNAS allele. tures. However, some recent reports identified patients A more recent study revealed a 19-kb genomic deletion who carry genetic and epigenetic defects associated with removing exon NESP55 and a large portion of the intron PHP 1b yet present with mild AHO features, particularly between NESP55 and antisense exon 4 in a kindred with the shortness of metacarpal bones.27,202,213-215 This may AD-PHP 1b with isolated loss of A/B methylation.206 suggest that Gsα imprinting occurs in more tissues than Since these patients lacked broad GNAS methylation currently recognized, though there are alternative expla- abnormalities, the identified deletion points to the exis- nations. Considering that individual AHO features can tence of another control element of A/B methylation, be observed in other disorders, the presence of AHO fea- without effects on other DMRs. The study of a mouse tures may be unrelated to the molecular genetic defects model in which Nesp55 transcription was prematurely underlying PHP 1b in these cases. In one case, the mother truncated revealed loss of imprinting at exon 1A (the of two affected siblings with short metacarpals and loss equivalent of human exon A/B) and, less consistently, the of A/B methylation also exhibited short metacarpals antisense promoter and exon XL.207 Taken together with despite lacking any GNAS epigenetic abnormalities,202 the genetic findings in PHP 1b patients, it appears that the suggesting that the finding of short metacarpals is unre- establishment of methylation imprints on the maternal lated to the epigenetic defect in that family. In addition, GNAS allele is essential for allowing expression of Gsα the observed coexistence of GNAS imprinting defects and in the proximal renal tubule and other tissues, including AHO can result from a large genomic deletion compris- the thyroid gland, in which this GNAS product is derived ing at least the promoter of Gsα and one or more differ- only from the maternal allele. Furthermore, the evidence entially methylated regions. In fact, such a large deletion that exon A/B methylation requires transcription from the leading to misdiagnosis of PHP 1b has been discovered in NESP55 promoter207 raises the possibility that even small a case with apparent loss of A/B methylation and AHO deletions or even point mutations can prevent the genera- features.216 tion of NESP55 pre-mRNA, thus leading to PHP 1b. Sporadic disease appears to be the most frequent cause PSEUDOHYPOPARATHYROIDISM TYPE 2 of PHP 1b. These cases all show broad GNAS methyla- tion defects. However, the maternal allele is frequently Dissociation regarding the impairment of PTH-induced shared between affected and unaffected siblings, suggest- nephrogenous cAMP formation and phosphaturia (i.e., ing that these cases may carry small de novo mutations in PHP 2) appears to be the least common form of PHP. this region. However, in some families the affected female Although typically sporadic, a case with familial form passed either allele #1 or allele #2 to their children, who of PHP 2 type has been reported,217 and several reports are all unaffected by PHP 1b and show no methylation describe evidence for a self-limited form of this disease abnormality at the GNAS locus.208,209 It is therefore possi- in newborns, which could indicate that it is transient in ble that some of the sporadic PHP 1b cases carry homozy- nature.35,218-220 The molecular defect and pathophysi- gous or compound heterozygous mutations in an entirely ologic mechanisms underlying this PHP variant remain different genomic location resulting in a putative auto- to be discovered. Because the defect underlying PHP 2 somal recessive mode of inheritance.208 Consistent with is associated with normal cAMP generation in response this conclusion, few patients affected by imprinting disor- to exogenous PTH, it was postulated that it is caused ders without obvious PTH-resistance show GNAS meth- by molecular defects that involve downstream of cAMP ylation changes.210,211 Moreover, methylation changes generation, such as protein kinase A.19 In fact, mutations 1158 PART 6 PARATHYROID GLAND, CALCIOTROPIC HORMONES, AND BONE METABOLISM

in the regulatory subunit of protein kinase A have been been described in more recent studies in which additional identified in some patients who show, in association with PRKAR1A mutations have been documented.44,45,231-233 characteristic skeletal abnormalities, biochemical defects Note that the distal tubular actions of PTH appear to be similar to those seen in PHP 2 (see later). Alternatively, impaired in patients with ADOHR, unlike in patients with the PTH signaling pathways that utilize other G proteins, PHP 1a and PHP 1b, consistent with the normal biallelic such as Gq or G11, may be defective in patients with expression of Gsα in the distal renal tubule. Neverthe- PHP 2. The signaling mediated by the Gq/G11 pathway less, the similarity between this disorder and PHP 1a with involves activation of phospholipase C, which in turn respect to the repertoire of hormone resistance is quite leads to the formation of second messengers inositol 1,4,5 striking and highlights the critical nature of the cAMP tris-phosphate (IP3) and diacylglycerol (DAG). This sig- signaling pathway in the actions of these hormones (Fig. naling pathway, which results in the stimulation of PKC 66-9).234 and an increase in intracellular calcium ions, was shown Genetic defects in another protein within the cAMP to be important in sustaining the phosphaturic actions on signaling pathway, cAMP phosphodiesterase type 4D PTH, as recently shown for mice expressing a PTHR-1 (PDE4D), have also been identified upon exome sequenc- mutant that fails to activate IP3/PKC signaling.221,222 ing of DNA from some patients with acrodysostosis.44,45 Serum calcium levels, which may affect the efficient utili- These heterozygous missense mutations in PDE4D are zation of intracellular calcium signaling pathways, appear associated with acrodysostosis, but the patients usually to be important for restoring PTH responsiveness in lack endocrine abnormalities. Other missense mutations PHP 2, as shown in some patients who normalized their of PDE4D have been identified in additional patients who phosphaturic response to PTH following normalization also show acrodysostosis in the absence of hormone resis- of serum calcium.223 It is also possible that the sodium- tance (termed acrodysostosis without hormonal resis- phosphate transporters in the proximal renal tubule are tance [ADOP4]).232,235 Thus, PDE4D mutations are not nonresponsive to PTH, thereby resulting in a defective associated with renal PTH resistance, and accordingly, phosphaturic, but not cAMP, response to exogenous unlike patients with PRKAR1A mutations, who demon- PTH. Such a defect, however, should preserve the action strate elevated basal urinary cAMP levels, patients with of PTH on 25(OH)D-1-α-hydroxylase and lead to normal PDE4D mutations show normal basal and PTH-induced 232 serum 1,25(OH)2D, unless it is combined with vitamin D urinary cAMP level. The biochemical mechanisms deficiency. Hypocalcemia as a result of vitamin D defi- underlying ADOP4 have yet to be elucidated. It is pre- ciency has also been associated with PTH resistance that dicted, however, that the PDE4D mutations are gain-of- entailed the phosphaturic effect of this hormone without function, given that PDE4D limits the intracellular level altering its potential to raise urinary cAMP,224 suggest- of cAMP.236 ing that some PHP 2 cases may in fact reflect vitamin D 225,226 deficiency. TREATMENT ACRODYSOSTOSIS WITH HORMONAL RESISTANCE The primary goal of treatment entails correction of abnormal serum biochemistries that result from PTH A recent report has described three patients who had and, in some cases, other hormone resistance, such as resistance to PTH and some other hormones but showed TSH resistance leading to hypothyroidism, which can be unimpaired urinary cAMP excretion in response to exog- treated by thyroid hormone replacement. GH deficiency enously administered recombinant PTH.227 Consistent can also be treated with recombinant human GH (rhGH) with resistance to the actions of PTH downstream of and is found to be efficacious in prepubertal patients with cAMP/PKA, PTH-induced urinary phosphate excretion PHP 1a.237 PPHP patients who carry de novo mutations was lost, and PTH-induced inhibition of urinary fractional on the paternal GNAS allele are often diagnosed late in calcium excretion was impaired. These unrelated patients, life (often when the children of female PPHP are diag- who could thus be classified as having a variant of PHP 2 nosed with PHP 1a), making it difficult to assess GH based on their renal PTH responsiveness, also displayed deficiency and thus the benefits of rhGH treatment in acrodysostosis, a skeletal dysplasia that resembles, but is PPHP patients. Clinical management of hypocalcemia in more severe than, the skeletal defects observed in AHO. patients with PHP is less difficult than in patients with Acrodysostosis, also known as Maroteaux-Malamut syn- hypoparathyroidism, because the distal tubular actions drome, includes severe bradydactyly, peripheral dysosto- of PTH in PHP patients are not impaired, providing sis, and nasal hypoplasia.228-230 This disorder is termed sufficient calcium reabsorption from the glomerular fil- acrodysostosis with hormonal resistance (ADOHR). trate. The treatment involves oral calcium supplements Analysis of DNA from these patients revealed a recur- and activated vitamin D analogues, such as 1,25(OH)2D rent heterozygous nonsense mutation of the gene encod- (calcitriol). Note that the active form of vitamin D is ing cAMP-dependent protein kinase (PKA) type 1 alpha required because of the lowered capacity of the proximal regulatory subunit (PRKAR1A; p.Arg368X). This muta- tubule to convert 25(OH)D into the biologically active tion leads to a mutant PRKAR1A protein missing the last 1,25(OH)2D. In addition, treatment of patients with 14 C-terminal amino acids. Accordingly, cAMP cannot PTH resistance should aim at keeping the serum PTH induce dissociation of this mutant regulatory subunit level within or close to the normal range rather than sim- from the catalytic subunit, thereby keeping PKA in its ply avoiding symptomatic hypocalcemia, since persistent inactive form. Several other patients with ADOHR have elevation of serum PTH will increase bone resorption 66 PSEUDOHYPOPARATHYROIDISM, ALBRIGHT’S HEREDITARY OSTEODYSTROPHY, AND PROGRESSIVE OSSEOUS 1159

Blomstrand’s disease

Adenylate PHP 1a and PHP 1b: Gsα cyclase hormonal resistance ± AHO β/γ

ATP cAMP AMP Acrodysostosis Regulatory without hormonal subunit Catalytic resistance (ADOP4) (PRKAR1A) subunit PDE4D

C R C R Cellular events R C R C

Inactive PKA Active cAMP PKA Acrodysostosis with hormonal resistance (ADOHR) 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.) and may eventually lead to hyperparathyroid bone dis- of the other gene products derived from GNAS. The ease.238,239 Due to intact PTH actions in the distal tubule, nature and the parental origin of the GNAS mutation is urinary calcium levels are usually low, and affected an important determinant of the clinical manifestations. individuals do not have a significant risk for developing Mutations that affect coding Gsα exons lead to broader kidney stones and nephrocalcinosis. In fact, during the clinical abnormalities than mutations that disrupt GNAS course of treatment, elevation of urinary calcium typi- imprinting. Due to the tissue-specific imprinting of Gsα, cally does not occur. Nevertheless, blood chemistries and hormone resistance develops only after maternal inheri- urinary calcium excretion in patients undergoing treat- tance. AHO typically occurs after both maternal and ment should be monitored annually, but more frequently paternal inheritance of coding Gsα mutations, although during pubertal development and once skeletal growth is the repertoire of some AHO features also follows an completed, as the requirements for treatment with cal- imprinted mode of inheritance. PPHP is used to describe cium and 1,25(OH)2D may need to be reduced. those patients with AHO who lack hormone resistance. AHO features vary markedly. An extreme manifesta- SUMMARY tion of AHO is progressive osseous heteroplasia, which appears to develop when Gsα mutations are present on PHP refers to a group of disorders characterized by PTH the paternal allele, possibly in combination with another resistance associated with hypocalcemia, hyperphospha- mutation. PHP 2 is rare, and the molecular determinants temia, and elevated serum PTH. Proximal tubular resis- of at least one variant with skeletal dysplasia involve the tance to PTH is the most prominent hormonal defect but, regulatory subunit of PKA. depending on the underlying genetic defect, resistance to other hormones is also observed. PTH and these other • For your free Expert Consult eBook with biblio- hormones all exert their actions via receptors that couple graphic citations as well as the ability to take notes, to Gsα. The primary genetic cause of PHP 1 and related highlight important content, search the full text, and disorders is mutations that affect the complex GNAS more, visit http://www.ExpertConsult.Inkling.com. locus, the gene encoding Gsα. These mutations result in decreased expression/activity of Gsα but also affect some REFERENCES 22. Huang CW, Chen YC, Tsai JJ. Paroxysmal dyskinesia with sec- ondary generalization of tonic-clonic seizures in pseudohypopara- 1. Albright F, Burnett CH, Smith PH, Parson W. Pseudohypo- thyroidism. Epilepsia. 2005;46:164–165. parathyroidism—an example of “Seabright-Bantam syndrome.” 23. Prashantha DK, Pal PK. Pseudohypoparathyroidism manifest- ­Endocrinology. 1942;30:922–932. ing with paroxysmal dyskinesias and seizures. Mov Disord. 2. Tashjian Jr AH, Frantz AG, Lee JB. Pseudohypoparathyroidism: 2009;24:623–624. assays of parathyroid hormone and thyrocalcitonin. Proc Natl 24. Kinoshita M, Komori T, Ohtake T, et al. 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