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PRIMARY HYPOPARATHYROIDISM

Primary hypoparathyroidism is caused by a group of heterogeneous conditions in which hypocalcemia and hyperphosphatemia occur as a result of deficient parathyroid hormone (PTH) secretion. This most commonly results from surgical excision of, or damage to, the parathyroid glands. However, autoimmune disease is also a significant factor in acquired cases, and genetic forms of hypoparathyroidism due to decreased PTH secretion are not uncommon (Table 1).

The signs and symptoms of hypoparathyroidism include evidence of latent or overt neuromuscular hyperexcitability due to hypocalcemia (Table 2). The effect may be aggravated by hyperkalemia or hypomagnesemia, but there is wide variation in the severity of the symptoms. Patients may complain of circumoral numbness, paresthesias of the distal extremities or muscle cramping which can progress to carpopedal spasm or tetany. Laryngospasm or bronchospasm and seizures may also occur. Other less specific manifestations include fatigue, irritability, and personality disturbance.

Severe hypocalcemia may be associated with a prolonged Q-T_c interval on electrocardiography, which reverses with treatment. More extensive cardiomyopathic changes are occasionally seen, particularly in adults. These include chest pain, elevated enzymes (CPK), left ventricular impairment, and T-wave inversion, suggestive of a myocardial infarction [1]. Patients with chronic hypocalcemia may have calcification of the basal ganglia or more widespread intracranial calcification, detected by skull X-ray or CT scan. Also seen are extrapyramidal neurological symptoms (more often with intracranial calcification), subcapsular cataracts, band keratopathy, and abnormal dentition.

Increased neuromuscular irritability may be demonstrated by eliciting a Chvostek or Trousseau sign. The Chvostek sign is a prolonged reflex contraction of the facial muscle in response to a digital tap on the cheek just anterior to the ear. As with other hyperreflexias, up to 20% of normal individuals may demonstrate a slight positive reaction. The Trousseau sign is carpopedal spasm induced by inflation of a blood pressure cuff covering the upper arm to 20 mm Hg above systolic blood pressure for three minutes. A positive response reflects the heightened irritability of nerves undergoing pressure ischemia.

In hypoparathyroidism, serum calcium concentrations are decreased and serum phosphate levels are increased. Serum PTH is low or undetectable. (The important exception is PTH resistance, discussed further below.) Usually, serum 1,25(OH)₂D is low, but alkaline phosphatase activity is normal. Despite an increase in fractional excretion of calcium, intestinal calcium absorption and bone resorption are both suppressed. The renal filtered load of calcium is decreased, and the 24-h urinary calcium excretion is reduced; nephrogenous cyclic AMP excretion is low and renal tubular reabsorption of phosphate is elevated. The Ellsworth-Howard test has been used to distinguish parathyroid resistance from glandular hypofunction. In patients with pseudohypoparathyroidism (see Table 3), parenteral administration of biologically active PTH fails to provoke an increase in plasma and urinary cyclic AMP and inorganic phosphate [2]. However, better biochemical profiling has led to a decline in the use of the test and it is now performed in only a few specialized centers. Decreased parathyroid gland reserve can be detected by an ethylenediaminetetraacetate (EDTA) infusion study [3], but this test is also restricted to metabolic centers where careful monitoring and expert observation are available.

The term "idiopathic hypoparathyroidism" has been traditionally used to describe isolated cases of glandular hypofunction when a cause is not obvious and there is no family history. However, hypoparathyroidism is a feature common to a variety of heritable syndromes that may present de novo. Hypoparathyroidism can occur because of a congenital hypoplasia/aplasia with or without other congenital anomalies such as dysmorphic facies, immunodeficiency, lymphedema, nephropathy, nerve deafness or cardiac malformation. Thus, in patients with hypoparathyroidism of uncertain onset, a careful examination of craniofacial features and assessment of endocrine, cardiac and renal systems should be performed to exclude a syndromic cause. Similarly, autoimmune hypoparathyroidism can occur as an isolated endocrine condition or with other glandular deficiencies in a pluriglandular autoimmune syndrome, requiring attention to multi-organ endocrine disfunction.

A significant number of patients with idiopathic hypoparathyroidism and hypercalciuria but no other anomalies may be found to have de novo activating mutations of the CASR gene. Because of the implications for treatment, molecular screening of patients with this condition may be warranted [4].

Familial isolated hypoparathyroidism (FIH) may show autosomal dominant, autosomal recessive, or X-linked inheritance. In a few instances of autosomal dominant disease, a mutation in the PTH gene has been found. In one family, a missense mutation in the signal sequence of the preproPTH precursor has been identified [5] and the resulting defect in processing of preproPTH to proPTH was thought to lead to reduced hormone production and chronic hypocalcemia, although it is not entirely clear why the remaining normal gene copy did not compensate for this under resting conditions. In one family with autosomal recessive hypoparathyroidism, a donor splice site mutation at the exon 2/intron 2 junction of the PTH gene (MIM# 168450 [6] - http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM) was identified [7]. The mutation leads to exon skipping and loss of exon 2 containing the initiation codon and signal sequence of preproPTH mRNA. In another family, a novel mutation in the signal sequence segregates with affected status. This mutation may prevent proper cleavage of the signal peptide during processing of the nascent protein [8]. In two multigeneration families with X-linked recessive hypoparathyroidism, the mutant gene has been localized to the distal end of the X chromosome (Xq26-27; MIM# 307700) [9-11]. A role for this gene in parathyroid gland development is suggested by the neonatal onset of hypocalcemia and the finding of parathyroid agenesis at autopsy in one of the patients.

Gain-of-function mutations in the calcium-sensing receptor (CASR) gene have been identified in several families clinically diagnosed with autosomal dominant hypocalcemia (ADH) (MIM# 601198), autosomal dominant hypoparathyroidism (MIM# 241400), and hypocalcemic hypercalciuria (MIM#146200) [12,13]. In the parathyroid gland, the activated CASR suppresses PTH secretion and in the kidney, it induces hypercalciuria which may contribute to the hypocalcemia. In many cases of ADH, the family history is positive, but de novo mutations are surprisingly common [4]. Mosaicism for de novo mutation in an otherwise healthy parent has been described [14], and may explain some cases of apparently recessive disease. More importantly, it has serious implications for counseling parents about the risk of recurrence.

More than fifty activating mutations (almost all missense) have been identified and appear almost equally divided between the amino-terminal third of the extracellular domain (ECD) and the transmembrane domain (TMD) (Figure 1). Of special interest is the cluster of six ECD mutations (A116T, N118K, L125P, E127A, F128L, and C129F) which cause an increase in receptor sensitivity to extracellular calcium, suggesting that this region is critical for receptor activation. This cluster overlaps the two cysteine residues –cys-129 and cys-131– putatively involved in the interface of the mature protein dimer [15]. Further details can be found in the locus-specific database –http://data.mch.mcgill.ca/casrdb/ [16].

Figure 1. Locations of CASR gene activating mutations found in patients with ADH. The schema outlines the relationship between CASR gene exons (II to VII) and the portions of the protein that they encode. Exons II to VI and the beginning of exon VII encode the ECD of ~610 amino acids, exon VII encodes the TMD of ~250 amino acids including membrane-spanning helices TM1-TM7 (indicated by the shaded boxes), ECL1 to ECL3, ICL1 to ICL3, and the ICD of ~200 amino acids. Recurrent mutations are highlighted. (Abbreviations: ECD, extracellular domain; TMD, transmembrane domain; TM, membrane-spanning helix; ECL, extracellular loop; ICL, intracellular loop; ICD, intracellular domain).

Recessively inherited FIH may occur with mutations of the glial cell missing-2 gene (GCMB; MIM# 603716). A patient with neonatal hypoparathyroidism was found to be homozygous for a partial deletion acquired from both parents [17], and a pair of siblings with homozygous mutations has been reported recently [18]. The GCMB gene localizes to chromosome 6p24.2 and appears to encode a transcription factor. It is expressed in the PTH-secreting cells of the developing parathyroid glands and is critical for their development in terrestrial vertebrates [19-21].

Hypoparathyroidism can occur as part of a 22q11 microdeletion, which has been identified as the common cause of the DiGeorge syndrome (MIM#188400) [22]. Deletions of 22q11 are also responsible for a spectrum of clinical conditions that include isolated congenital heart disease and velocardiofacial (VCF) syndrome [23]. With DiGeorge syndrome, patients may present with neonatal hypocalcemic seizures due to hypoparathyroidism, severe infections due to thymic hypoplasia, and conotruncal heart defects [24]. However, it is not unusual for the hypoparathyroidism to remain asymptomatic until adolescence or require provocative testing to confirm the decreased parathyroid reserve in normocalcemic adults [25]. Craniofacial abnormalities include cleft palate, pharyngeal insufficiency and mildly dysmorphic facies. In the VCF syndrome, anatomical anomalies of the pharynx are prominent and hypernasal speech due to abnormal pharyngeal musculature with or without cleft palate is typical. In most patients, some degree of intellectual deficit is present and there is strong predisposition to psychotic illness (schizophrenia or bipolar disorder) in adolescents and adults [26]. Further information, both clinical and educational, can be found at various web sites devoted to this condition (eg., http://www.vcfsef.org/).

The syndrome complex arises from a failure of the third and fourth pharyngeal pouches to develop, leading to agenesis or hypoplasia of the parathyroid glands and thymus. Because a microdeletion is involved, the identification of novel developmental genes in the 22q11 region is being keenly pursued. Mouse models with Tbx1 transcription factor haploinsufficiency have been used to establish the essential contribution of this factor to conotruncal development [27]. It is likely, however, that full expression of DiGeorge syndrome involves loss of other contiguous genes [28]. Although many cases of DiGeorge syndrome occur de novo, autosomal dominant inheritance is not unknown. In utero influences may be important determinants of the clinical picture, since there are at least three reported instances of monozygotic twins with discordant phenotypes [29-31]. Phenocopies occur with diabetic embryopathy, fetal alcohol syndrome, and retinoid embryopathy.

The clinical features of this condition also occur with other cytogenetic abnormalities, notably chromosome 10p haploinsufficiency [32,33]. Hypoparathyroidism is part of the Barakat or HDR (Hypoparathyroidism, nerve Deafness, and Renal dysplasia) syndrome (MIM # 146255) [34,35]. Deletions of two non-overlapping regions of chromosome 10p contribute to a DiGeorge-like phenotype (the DiGeorge critical region II on 10p13-14 [21,36]) and the HDR syndrome (10p14-10pter [37,38]). Deletion mapping studies in two HDR patients defined a region containing the GATA3 gene which encodes a zinc finger transcription factor involved in vertebrate embryonic development. GATA3 haploinsufficiency and loss-of-function mutations of this gene have been reported in other HDR patients [39,40]. Thus, GATA3 appears essential for normal embryonic development of the parathyroids, auditory system, and kidney.

In another congenital disorder, Kenny-Caffey syndrome, hypoparathyroidism is found variably associated with the typical picture of growth retardation, osteosclerosis, cortical thickening of the long bones, and delayed closure of the anterior fontanel [41,42]. Both dominant and recessive modes of inheritance have been observed (MIM# 127000 and MIM# 244460, respectively). The Sanjad -Sakati syndrome (MIM# 241410) is a similar recessive condition characterized by congenital hypoparathyroidism, seizures, growth and developmental retardation and characteristic dysmorphic features, including deep set eyes, depressed nasal bridge with beaked nose, long philtrum, thin upper lip, micrognatia and large, floppy ear lobes. Radiographs show medullary stenosis reminiscent of Kenny-Caffey syndrome [43]. Linkage studies have localized recessive Kenny-Caffey and Sanjad-Sakati syndromes to 1q42-43, and an international consortium has reported causative mutations in a tubulin chaperone protein, TBCE [44,45].

Hypoparathyroidism is also a variable component of the neuromyopathies caused by mitochondrial gene defects. Among these are the Kearns-Sayre syndrome (ophthalmoplegia, retinal degeneration, and cardiac-conduction defects) (MIM# 530000), the Pearson marrow pancreas syndrome (lactic acidosis, neutropenia, sideroblastic anemia, and pancreatic exocrine dysfunction) [46] (MIM# 557000) and mitochondrial encephalomyopathy (MIM# 540000). The molecular defects range from large deletions and duplications of the mitochondrial genomes in a large number of tissues [47,48] to single base-pair mutations in one of the transfer RNA genes found only in a restricted range of cell types (MIM# 590050). The role of these mitochondrial mutations in the pathogenesis of hypoparathyroidism remains to be clarified, but hypomagnesemia due to a renal wasting can present with symptoms similar to hypocalcemia and the two may co-exist in these disorders.

Long-chain hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency (MIM# 600890) is an inborn error of oxidative fatty acid metabolism that may be accompanied by hypoparathyroidism [49]. Whether the parathyroid disease is directly related to the enzyme deficiency or secondary to the accompanying mitochondrial disease needs further study.

Parathyroid insufficiency and symptoms of hypocalcemia are occasionally seen in inherited metabolic disorders leading to excess storage of iron (thalassemia, Diamond-Blackfan anemia, hemochromatosis) or copper (Wilson disease). In most instances, there is similar dysfunction in other endocrine glands, and the parathyroid disease is usually mild. Nonetheless, recognition of the hypoparathyroid state may help explain otherwise non-specific symptoms and aid in overall management of these multisystem diseases.

Autoimmune parathyroid gland ablation or destruction

Antibodies directed against parathyroid tissue have been detected in over 30% of patients with isolated hypoparathyroid disease, and over 40% of patients having hypoparathyroidism combined with other endocrine deficiencies. Antibodies against the extracellular domain of the parathyroid CASR were reported in more than half of patients with either type 1 autoimmune polyglandular syndrome (APS-1, also known as autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy or APECED, MIM# 240300, [50]) or acquired hypoparathyroidism associated with autoimmune hypothyroidism [51]. This finding was confirmed in a subsequent study of 51 cases of idiopathic hypoparathyroidism, but there was a 13% positive rate in controls [52]. However, two large studies of APS-1 patients failed to reveal a significant preponderance of positives [53, 54]. Although some have argued that CASR antibody assays are clinically indicated in acquired hypoparathyroidism [55], others have demurred, and it remains to be seen whether the autoantibodies are of primary or secondary importance [56].

In APS-1, the most common associated manifestations are hypoparathyroidism with mucocutaneous candidiasis and Addison's disease. Additional features include pernicious anemia, chronic active hepatitis, alopecia, keratitis, gonadal failure, thyroid disease, pancreatic insufficiency and diabetes mellitus [50]. The phenotype is highly variable and patients may not express all elements of the basic triad, leading to the suggestion that the criteria used for molecular screening be relaxed [57]. The disease usually presents in infancy with candidiasis, followed by hypoparathyroidism in the first decade, and then adrenocortical failure in the third. Interestingly, there is nearly 100% penetrance of hypoparathyroidism in females, but less than 60% in males, even though the adrenal hypofunction affects both sexes equally [54]. Moreover, patients who develop the adrenal hypofunction first are less likely to be male and may never develop hypoparathyroidism. The responsible gene, called the autoimmune regulator (AIRE), maps to chromosome 21q22 and encodes a putative transcriptional regulator [58-60]. Showing either sporadic or autosomal-recessive inheritance, APECED has been associated with more than 40 different mutations of the AIRE gene, and updates can be found in the online mutation database (http://bioinf.uta.fi/AIREbase/).

Several clinical disorders characterized by end-organ resistance to PTH have been described collectively by the term pseudohypoparathyroidism (PHP) [61-64]. They are associated with hypocalcemia, hyperphosphatemia, and increased circulating PTH, but target tissue unresponsiveness to the hormone manifests as a lack of increased cAMP excretion in response to PTH administration [65]. The biochemical characteristics of these disorders are contrasted with those of hypoparathyroidism in Table 3.

Fuller Albright first recognized that the likely cause of the hypoparathyroid state in PHP is a constitutive absence of target tissue responsiveness [66]. In many patients, the end-organ resistance is accompanied by a specific pattern of physical findings, called Albright hereditary osteodystrophy (AHO; MIM#300800). Typically patients have short stature, round facies, brachydactyly, obesity, and ectopic soft tissue or dermal ossification(s) (osteoma cutis) (Figure 2). In the calvaria, this may manifest as hyperostosis frontalis interna [61]. Intracranial calcification(s), cataracts and band keratopathy, subcutaneous calcifications, and dental hypoplasia are also common but are likely the consequences of longstanding hypoparathyroid hypocalcemia (Table 4, see below figure 2). The brachydactyly may be asymmetric or not, and may involve one or both hands or feet, but the pattern is quite distinctive. The shortening tends to involve the first distal phalanx, with a thumbnail (or first toenail) that is wider than it is long. The fourth and fifth metacarpals (or metatarsals) are frequently shortened out of proportion to the others and the second metacarpal is often spared. Radiographic analysis of the hands (pattern profiling) may be helpful in assessment of the brachydactyly (Figure 2)[65].

Figure 2. Features of Albright Hereditary Osteodystrophy (AHO). [A] Young woman with short stature (~ 3rd centile), disproportionate shortening of the limbs, generalized obesity, and round, flattened face. [B] Radiograph of the hand showing the shortened 4th and 5th metacarpals. [C] Fist with the characteristic 'dimples' over the 3rd, 4th, and 5th digits replacing the knuckles formed by the distal head of normally sized metacarpal bones (Archibald sign). [D] Brachydactyly of the hand, with the short 4th and 5th digits, the greatly foreshortened terminal 1st digit, and very short, wide thumbnail (potter's thumb). (Reproduced from Levine, 2000, with permission).

Although affected patients are generally short as adults, their bone age as children may be advanced and growth accelerated [67]. In patients who retain PTH responsiveness in bone, signs of hyperparathyroid bone disease (osteitis fibrosa cystica) may be observed, complicating the picture [68,69]. In infants and young children, rachitic changes are not uncommon. The round facies, short neck, and low, flat nasal bridge are often accompanied by central obesity, which varies considerably in extent, with or without accompanying hypothyroidism. Patients with AHO are probably predisposed to hypertension [70], conductive and sensorineural hearing loss [61,71], cord compression due to spinal anomalies [72], and movement disorders due to basal ganglial calcification [73]. Anosmia was thought to be associated with loss of G-protein olfactory receptor function, but this does not seem to be true in all cases [74]; it is not a problem for patients and is of limited clinical usefulness [61].

In most PHP1a patients, particularly, the features of AHO may be subtle in infancy or early childhood; in a few, there is little to see even in adulthood. Moreover, patients with brachydactyly, mental retardation, and other features closely resembling AHO, but with normal Gsalpha expression, have been found to carry microdeletions of chromosome 2q27 [75]. Genes important for skeletal and neurological development probably lie within this region, but 2q27 deletion patients have no endocrine abnormalities.

PHP1a patients, characterized by AHO, PTH resistance, and evidence of target organ resistance to other hormones, are usually found to have a reduction in the activity of the Gs alpha subunit, which is part of the membrane associated G-protein complex - transducing signals between G-protein coupled receptors and adenylate cyclase [76-78].

The GNAS1 gene encoding the Gs alpha protein maps to 20q13.2-13.3 and has at least 4 alternative transcriptional start sites (Figure 3) and an antisense transcript, NESPas [79]. The three upstream exons are genetically imprinted in a way that affects penetrance of the PHP phenotype. The marked excess of maternal transmission in PHP1a with AHO [80] can be explained by imprinting suppression of the paternal exon 1 utilization which would otherwise allow expression of the normal paternal Gs alpha [81]. Full expression of a coding GNAS1 mutation, which occurs in maternally transmitted cases, leads to AHO plus hormone resistance (PHP1a), whereas AHO alone (pseudopseudohypoparathyroidism or PPHP) is observed with a paternally transmitted mutation. Despite clinical evidence supporting imprinting in portions of the kidney tubule, it has been difficult to confirm this experimentally in humans [82]. The imprinting is complex and involves multiple differentially methylated regions (DMR) [79]. Moreover, it is tissue-specific and may vary with developmental stage [83,84], although key imprinting of the XL alpha S DMR is a primary event that occurs during gametogenesis and is maintained thereafter [85]. Ablation of the Gs alpha ortholog in mice (Gnas) has confirmed that maternal transmission of the deleted allele results in PTH resistance but paternal transmission does not. The homozygous deletion is an embryonic lethal [86].

Figure 3. Simplified view of the GNAS1 region and its transcripts. The normal allele-specific methylation and expression patterns of the four alternate first exons of GNAS1 which splice onto exon 2 to produce transcripts encoding NESP55, XL alpha s, a transcript of unknown function (exon 1A), and Gs alpha (exon 1). NESP55 and XL alpha s promoters are oppositely imprinted: NESP55 is expressed from the maternal allele and its promoter region is methylated on the paternal allele, whereas XL alpha s is expressed from the paternal allele and its promoter is methylated on the maternal allele. Exon 1 is probably paternally imprinted in some tissues e.g., renal proximal tubule cells, indicated by the dashed arrow. NESP55 protein is unrelated to Gs alpha, and its entire coding region is located within its first exon. In contrast, XL alpha s and Gs alpha proteins have identical COOH-terminal domains (encoded by exons 2-13), while their unique NH2-terminal domains are encoded within their respective first exons. Exon 1A does not have a translational start site, but is transcriptionally active. Loss of exon 1A imprinting (methylation) leads to decreased Gs alpha expression in renal proximal tubules and some other hormonal tissues, and is the usual cause of PHP1b. (From Liu et al., 2000, with permission).

A variety of inactivating mutations in the portion of the GNAS1 gene encoding Gs alpha have been identified in PHP1a patients [87-91]. The spectrum includes missense mutations, point mutations impairing efficient and accurate splicing, and small insertion/deletion mutations. The 4bp deletion in exon 7 (deltaGACT 188/190) has been observed in multiple unrelated cases, suggesting that this may be a hot spot [63,91]. Several other mutations have also been observed in more than one kindred, indicating that additional susceptibility regions may exist. The identification of de novo germline mosaicism [92] is consistent with the view that most sporadic cases harbour new mutations, but the separation of such sporadic cases from familial ones, in which there is suppression of phenotype due to imprinting, may be difficult without detailed molecular studies.

PHP1a cases have been described in which no mutations of the GNAS1 gene have been found. This may be because the mutation is in a regulatory region of the gene not yet examined, or it may be that PHP1a can arise from more than one type of defect in PTH signaling.

Typical PHP1a is associated with multiple hormone resistance, including thyroid stimulating hormone (TSH) and gonadotropins, causing hypothyroidism and gonadal failure, respectively. Because the hypothyroidism may express before hypocalcemia is observed [63], early surveillance of thyroid function is warranted. However, thyroid replacement from birth does not appear to prevent the mental deficit typical of PHP1a. In women, the hypogonadism is partial [93] so that oral contraceptives may help regulate the menstrual cycle. Estrogen can antagonize bone resorption leading to an exacerbation of hypocalcemia [94], but placental 1,25-dihydroxyvitamin D synthesis likely obviates this effect altogether in pregnancy so women are frequently normocalcemic at that time [95]. Abnormalities of the somatotropin axis have also been reported, with documentation of subnormal growth hormone release following stimulation by L-arginine or growth hormone releasing hormone [96,97]. Whether replacement therapy would be beneficial remains uncertain, since those with paternally derived mutations and apparent absence of hormone resistance have the AHO phenotype with short stature and obesity [98].

PHP1b is not associated with AHO or a generalized reduction in Gs alpha expression [62]. PHP1b patients show a defect in renal PTH signaling, but an apparently normal response to PTH in bone. Affected individuals show normal skeletal architecture and development but are functionally hypoparathyroid. Although biochemical abnormalities of thyroid hormone regulation may be seen, and abnormalities of uric acid metabolism have been documented [99], clinically significant hormone resistance is restricted to PTH alone. Although it was thought at one time that these findings could be explained by a defect in the parathyroid receptor (PTHR1, MIM#168468), sequencing in PHP1b patients show no mutations in protein-coding exons or gene promoter regions of the gene [100-102] and studies of PHP1b families show no linkage to PTHR1 [103,104].

In four PHP1b kindreds, linkage to chromosome 20q13.3 was established, the same region which includes the GNAS1 locus [103]. In these families, the pattern of transmission suggests paternal imprinting (Figure 4) and inheritance is therefore the same as for PHP1a. Liu et al. [81] studied 13 PHP1b subjects, some of whom had bone responsiveness to PTH. All lacked methylation of the alternate exon 1A, which is postulated to inhibit expression of the functional exon 1-containing Gs alpha transcript in renal tissues only (Figure 3). Thus the loss of methylation suppression of the maternal exon 1A allele leads to the silencing of the maternal as well as paternal exon 1 allele, causing PTH resistance specifically in renal proximal tubule cells. Furthermore, a PHP-1b patient with paternal uniparental isodisomy caused by maternal deletion of a chromosome 20q segment has been described [105], emphasizing the importance of a normal maternal allele for renal PTH responsiveness. Genetic analysis indicated that mutations in a regulatory region some distance from the GNAS1 coding exons were likely to account for the unique imprinting defect(s) associated with PHP1b [106]. A search for the mutation, presumably in a cis-acting element close to 20q13.3, revealed the presence of a common 3kb microdeletion that segregated with the disease in 12 kindreds with familial PHP1b and also occurred in 4 sporadic cases, presumably de novo [107]. The deletion, flanked by repeats, includes 3 exons of the STX16 gene which encodes for syntaxin-16 and is not itself imprinted. In another two other PHP1b kindreds, deletions of the NESP55 DMR segregated with the disease [108]. In this instance, however, the 1A DMR was not the only region to lose the differential methylation required to allow maternal expression of Gs alphas in the kidney. Maternal methylation was also lost in the regions of the XL alpha s and NESPas promoters, suggesting a more widespread epigenetic defect. Neither transcript is predicted to have physiological effects, and it seems likely that the phenotypes will be similar.

A PHP1b family with a novel Gs alpha mutation, deletion of isoleucine-382 in the carboxyl terminus (leading to uncoupling from the PTHR and isolated PTH resistance), shows transmission through 3 generations, consistent with paternal imprinting [109]. However, such mutations within Gs alpha coding exons are probably rare [81].

Figure 4. Laboratory findings, clinical features and haplotyping with 20q13 markers for a large family with PHP1b. The grandmother is clinically normal (hatched symbol), but 6 of 12 offspring have hypocalcemia and elevated circulating PTH levels (solid symbols). Two second-generation offspring of affected females are likewise affected, but another two children of affected males who bear the same set of GNAS1-linked markers on 20q13 are unaffected. Accurate genetic counselling for second- and third-generation members of this pedigree is therefore enhanced by the molecular studies. Important also for this family is the knowledge that PHP1b is not associated with AHO and its accompanying neurodevelopmental and connective tissue complications. (Reproduced from Juppner et al., 1998, with permission).

PHP1c and PHP2Patients with PHP1c have multiple hormone resistance but normal Gs alpha activity. The defect may be in other components of the receptor-adenylate cyclase system, such as the catalytic unit, but there is at least one example of a GNAS1 defect (p.T391X [110]). Finally, patients with PHP2 have a normal urinary cAMP response to PTH but an impaired phosphaturic response [111]. The defect could be in PKA, one of its substrates or targets, or in a component of the PTH-PKC signaling pathway.

Other Phenotypes Associated with GNAS1 MutationsIn contrast to the PHP phenotype associated with inactivating GNAS1 mutations, a different form of sporadic bone disease, (polyostotic) fibrous dysplasia, results from de novo GNAS1 mutations that are activating [112]. A more severe form of this disease (panostotic fibrous dysplasia) with hyperphosphatasia and hyperphosphaturic rickets, is also known [113,114]. A missense mutation in exon 13 (A366S) results in a Gs alpha protein that is unstable at 37°C [115], but constitutively active at lower temperatures. Affected patients have PHP due to PTH resistance and precocious puberty (testitoxicosis) due to hormone-independent constitutive activation of luteinizing hormone receptors at lower ambient temperatures in the testes [54]. Undoubtedly, other patterns of hormone-receptor interaction due to GNAS1 mutation await discovery. Similarly, identification of mutations in patients with congenital osteoma cutis and severe heterotopic ossification syndromes suggest that these connective tissue conditions are another variant in the phenotypic spectrum of GNAS1-related disease [116-118].

Differential diagnosis and genetic counselingPatients with dysmorphic features resembling AHO may require careful endocrinologic work-up to confirm and delineate the form of PHP that is present and rule out the possibility of a phenocopy due to a 2q microdeletion. Similar studies of family members may also be warranted, since the biochemical and clinical features vary within families. If PHP1a with AHO is established, genetic counselling may aid in understanding the multisystemic nature of the disorder, particularly in relation to the patient's growth and development, and later-onset connective tissue complications. In this context, the distinction between PHP1a and PHP1b is important, since the latter does not include AHO, nor its associated physical features or cognitive deficit. For either PHP1a or PHP1b, however, extensive counselling may be required to adequately explain the various implications of paternal imprinting for the parent-specific recurrence risks in offspring. Germline mosaicism has been reported [92], which is clearly important in assessing risks for recurrence in future sibs of a singleton family. Molecular testing may help clarify and validate the inheritance pattern in any given family.

The type 1 parathyroid hormone receptor (PTHR1 - MIM#168468) is a plasma membrane receptor of the G-protein family that interacts with GNAS. It responds to two ligands, PTH and the PTH-related peptide (PTHRP) [119]. It would thus be predicted that deleterious mutations might show resistance to PTH, as well as evidence for a defect of PTHrP action. Indeed, humans heterozygous for inactivating mutations in the PTHR1 gene are normal, whereas homozygosity manifests as Blomstrand lethal chondrodysplasia (MIM#215045), a recessive short-limbed dwarfism with craniofacial malformations, hydrops, hypoplastic lungs and aortic coarctation [120-123]. The bones show accelerated endochondral ossification and deficient remodeling, in keeping with the regulatory role that PTHR1 plays in bone formation in utero. A milder form of recessively inherited skeletal dysplasia, known as Eiken syndrome (MIM#600002), has also been linked to mutations of PTHR1 [124], suggesting that a wider range of skeletal phenotypes can be attributed to this gene.

A PTHR1 mutation (c.448C>T predicting p.R150C) was identified in two out of six patients with enchondromatosis (Ollier's disease - MIM#166000), a familial disorder with evidence of autosomal dominance characterized by multiple benign cartilage tumors, and a predisposition to malignant osteocaroma [125]. A follow-up study of 31 patients failed to reveal any PTHR1 mutations [126], and it is likely that the condition is genetically heterogeneous.

In humans, hypomagnesemia leads to a suppression of parathyroid hormone release and some degree of peripheral resistance. Although the exact molecular mechanism underlying the suppression of the parathyroid gland in hypomagnesemia is unknown, it is important to recognize that laboratory testing in cases of hypocalcemia with reduced PTH should include measurement of serum magnesium, particularly in newborns [127].

An unusual state of parathyroid resistance in utero may be seen in infants with I-cell disease or mucolipidosis II (MIM#252500) [128]. Late-trimester radiographs or ultrosonographs may show skeletal changes typical of severe hyperparathyroidism, including demineralization and long bone fracture, periosteal cloaking. At birth, serum calcium is normal or decreased, and serum PTH is greatly elevated. The newborns show clinical features typical of a severe mucopolysaccharidosis, such as gingival hypertrophy, coarse facial features, and failure to thrive. Over the ensuing months the hyperparathyroid profile abates but the skeletal abnormalities become more typical of the dysostosis that characterizes a number of MPS disorders. Vitamin D therapy has been used to correct the hypocalcemia, but the hyperparathyroid-resistance state may be mild and self-limiting. The I-cell disease itself is progressive and untreatable, leading to death before the second decade.

The goal of treatment in hypoparathyroid states is to raise the serum calcium sufficiently to alleviate acute symptoms and prevent the complications of chronic hypocalcemia [129] . The calcium concentration required for this purpose is generally the low-normal range. Acute or severe symptomatic hypocalcemia is best treated with intravenous calcium infusion. Initial doses of 2 to 5 millimoles of elemental calcium as the gluconate salt can be given over a 10 to 20 minute period, followed by 2 millimoles elemental calcium per hour as a maintenance dose, to be adjusted according to symptoms and biochemical response. Care must be taken to ensure that the infusion does not extravasate, and ionized or total calcium levels should be monitored frequently on a stat basis. Doses in children 5 to 14 years of age need to be adjusted for body weight, while neonates and infants require age-specific dosing schedules. In adults, intravenous vitamin D therapy is not needed. Hyperphosphatemia, alkalosis and hypomagnesemia should be corrected concomitantly if present, and if a patient is receiving a digitalis-like drug, electrocardiographic monitoring should be performed to prevent digitalis toxicity which can be precipitated by the rise in serum calcium. Post-surgical hypocalcemia is now rarely severe and usually transient with appropriate management [130], but longterm effects remain a concern [131].

The mainstay of chronic treatment is oral calcium and vitamin D, which should be started as soon as possible to allow reduction and discontinuation of the intravenous calcium. Oral calcium comes in several forms, but calcium carbonate is generally the least expensive. A total of 20 to 80 millimoles elemental calcium daily (2 to 8 g calcium carbonate per day) is generally effective, but should be given in divided doses and adjusted on the basis of gastro-intestinal tolerance, relief of hypocalcemic symptoms, and appropriate biochemical response. Vitamin D is preferably administered as calcitriol (0.25 to 0.5 micrograms per day) but, if cost is a factor, pharmacological doses of cholecalciferol or ergocalciferol or calcidiol may be less expensive and equally efficacious [132] . Cholecalciferol and ergocalciferol have the longest duration of action and can result in sustained toxicity. It is therefore appropriate to institute a starting dose of 25,000 IU/day and titrate upwards (to 100,000 IU/daily) with assessment of serum and urinary parameters afterwards with follow-up at 6 and 12 months, even if the patient is relatively asymptomatic. In any event, serum calcium and 24 hour urinary excretion should be carefully monitored when therapy is started and continued until the patient is stabilized. Hypercalciuria that occurs as treatment is initiated, even prior to the normalization of the serum calcium, may warrant an ongoing assessment of nephrocalcinosis, most sensitively detected by renal ultrasound. Consequently, only a low-normal serum calcium may be attainable, but many patients feel well enough that there is no need to entirely normalize the serum calcium. That way, the risk of renal failure due to chronic hypercalciuria – especially problematic in patients with CASR activating mutations [4] – is minimized. Nevertheless, a significant number of patients report problems with easy fatigue and exhaustion, and mood disturbances (e.g, depression, anxiety, hostility, and paranoid ideation) not in keeping with the degree of hypercalcemia, suggesting that there may be non-calcitropic effects of PTH not remedied by maintenance of normocalcemia alone [131]. Preliminary studies suggest that hypercalciuria is less of a problem with daily subcutaneous injections of synthetic human PTH [133], but widespread trials have not been reported. Calcilytics – drugs that antagonize the calcium-sensing receptor and promote PTH secretion – are a promising alternative for disorders with intact but hypofunctioning parathyroid glands [134].

If the serum calcium attainable with oral calcium and calcitriol is below the normal range and the patient remains symptomatic, then a trial of a thiazide diuretic may be considered, with the aim of reducing the hypercalciuria to raise the serum calcium further. The argument for efficacy seems greatest for responsive forms of autosomal dominant hypocalcemia due to activating CASR mutations, since the thiazide-sensitive transporter, SLC12A3 (MIM#600968), is a downstream target suppression by activated CASR in the kidney. For reasons that are not clear, however, thiazides work well in some patients [135] but not others.

As the serum calcium is normalized, elevated serum phosphate concentrations generally decline but phosphate-binding gels such as aluminum hydroxide are occasionally helpful in reducing hyperphosphatemia at the beginning of therapy.

In patients with intracranial calcifications, patients may experience seizures related to chronic neuropathic changes and it may be necessary to add appropriate anti-epileptic medication(s). In all chronically hypocalcemic patients, ocular assessments should be performed periodically.