Diagnosis of asymptomatic primary hyperparathyroidism: proceedings of the Fourth International Workshop.

S P E C I A L

F E A T U R E

C o n s e n s u s

S t a t e m e n t

Diagnosis of Asymptomatic Primary Hyperparathyroidism: Proceedings of the Fourth International Workshop Richard Eastell, Maria Luisa Brandi, Aline G. Costa, Pierre D’Amour, Dolores M. Shoback, and Rajesh V. Thakker Academic Unit of Bone Metabolism (R.E.), University of Sheffield, Sheffield S5 7AU, United Kingdom; University of Florence (M.L.B.), 50133 Florence, Italy; Department of Medicine (A.G.C.), Division of Endocrinology, Metabolic Bone Diseases Unit, College of Physicians and Surgeons, Columbia University, New York, New York 10032; Department of Medicine (A.G.C.), Division of Endocrinology, São Paulo Federal University, São Paulo 04021-001, Brazil; Centre Hospitalier de l’Université de Montréal (P.D.), Hôpital St-Luc and Department of Medicine, University of Montréal, Montréal, Québec, Canada H3C 3J7; Endocrine Research Unit (D.M.S.), San Francisco Department of Veterans Affairs Medical Center, University of California, San Francisco, California 94121; and Academic Endocrine Unit (R.V.T.), Radcliffe Department of Medicine, University of Oxford, Oxford OX3 7LJ, United Kingdom

Objective: Asymptomatic primary hyperparathyroidism (PHPT) is a common clinical problem. The purpose of this report is to provide an update on the use of diagnostic tests for this condition in clinical practice. Participants: This subgroup was constituted by the Steering Committee to address key questions related to the diagnosis of PHPT. Consensus was established at a closed meeting of the Expert Panel that followed. Evidence: Each question was addressed by a relevant literature search (on PubMed), and the data were presented for discussion at the group meeting. Consensus Process: Consensus was achieved by a group meeting. Statements were prepared by all authors, with comments relating to accuracy from the diagnosis subgroup and by representatives from the participating professional societies. Conclusions: We conclude that: 1) reference ranges should be established for serum PTH in vitamin D-replete healthy individuals; 2) second- and third-generation PTH assays are both helpful in the diagnosis of PHPT; 3) normocalcemic PHPT is a variant of the more common presentation of PHPT with hypercalcemia; 4) serum 25-hydroxyvitamin D concentrations should be measured and, if vitamin D insufficiency is present, it should be treated as part of any management course; 5) genetic testing has the potential to be useful in the differential diagnosis of familial hyperparathyroidism or hypercalcemia. (J Clin Endocrinol Metab 99: 3570 –3579, 2014)

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his report focuses on the diagnosis of primary hyperparathyroidism (PHPT). The key questions addressed are: Question 1: A, Do third-generation PTH assays perform better clinically than second-generation PTH assays for the diagnosis of PHPT? B, Do we now have optimal reference intervals for

serum PTH, and are these intervals based on individuals who are vitamin D replete? C, Is normocalcemic hyperparathyroidism a part of the diagnostic spectrum of PHPT? Question 2: A, Should we measure 25-hydroxyvitamin D [25(OH)D] in all patients with suspected PHPT? B,

ISSN Print 0021-972X ISSN Online 1945-7197 Printed in U.S.A. Copyright © 2014 by the Endocrine Society Received February 11, 2014. Accepted May 22, 2014. First Published Online August 27, 2014

Abbreviations: BMD, bone mineral density; CASR, calcium-sensing receptor; CDK, cyclindependent kinase; eGFR, estimated glomerular filtration rate; FHH, familial hypocalciuric hypercalcemia; FIHPT, familial isolated hyperparathyroidism; HPT-JT, hyperparathyroidismjaw tumor; MEN, multiple endocrine neoplasia; NPHPT, normocalcemic PHPT; N-PTH, amino-terminal PTH; 1,25(OH)2D, 1,25-dihydroxyvitamin D; 25(OH)D, 25-hydroxyvitamin D; PHPT, primary hyperparathyroidism; UCCR, urinary calcium:creatinine clearance ratio.

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J Clin Endocrinol Metab, October 2014, 99(10):3570 –3579

doi: 10.1210/jc.2014-1414

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For the diagnostic evaluation of PHPT, second- and third-generation PTH assays perform similarly and are better than the first-generation assay (1). The second-generation PTH assay, also known as the intact PTH assay, utilizes an improved technology with greater specificity compared to the first-generation assays (Figure 1). It recognizes intact PTH (1– 84) but was also shown to crossreact with large amino-truncated Figure 1. Circulating PTH molecular forms. Role of three generations of PTH assays. The firstPTH fragments, of which the most generation assays are mainly detecting C-terminal fragments. The-second generation assays are abundant is PTH (7– 84). The thirdmainly detecting intact PTH and some N-PTH and some non (1– 84) PTH. The third-generation generation assay known as the assays are mainly detecting PTH and some N-PTH. “whole” PTH assay was later develHow should the reference ranges for different assays be oped to be more specific for PTH (1– 84) using a label interpreted? C, What represents the threshold for over- antibody directed at PTH (1– 4), and thus preventing treatment? D, Is it useful to measure 1,25-dihydroxyvita- cross-reactivity with PTH (7– 84) fragments. However, in min D [1,25(OH)2D] in patients with PHPT, and under addition to the whole PTH (1– 84) molecule, the thirdwhat circumstances? generation assay detects a post-translational modified Question 3: A, What is the genetic basis for syndromic form of PTH (1– 84) in region 15–20 by phosphorylation and nonsyndromic forms of PHPT? B, What is the value of of a serine residue known as nontruncated amino-terminal genetic testing in clinical practice? C, What should be the PTH (N-PTH), which has been shown to represent up to clinical approach to gene testing in a patient with 10% of PTH values in normal individuals and 15% in hypercalcemia? advanced renal failure. Furthermore, N-PTH can be overAn electronic literature search was conducted using produced in specific situations such as parathyroid carciPubMed on all published literature between June, 2008, noma and severe hyperparathyroidism (2). Nonetheless, and August, 2013. Keywords were combined to identify the mean PTH concentrations with the third-generation the relevant articles. Manuscripts focusing on the key assay are typically lower than with the second-generation questions were prepared by the team members. These assay. manuscripts were presented at the Fourth International Studies that compare second- and third-generation Workshop on the Management of Asymptomatic Primary PTH assays with regard to diagnostic sensitivity of PHPT Hyperparathyroidism on September 19 –21, 2013, in are few. Only four published studies are available (3– 6), Florence, Italy. After the presentations and discussions, a all of which preceded the last PHPT workshop. We conconsensus panel convened, and each question was disclude, as we did in the proceedings of the last workshop cussed in detail. Through discussion and dialogue, con(1), that the diagnostic sensitivity for PHPT is similar besensus was achieved. The recommendations presented in tween second- and third-generation PTH assays (Table 1). this document reflect the opinion of the panel. It is important to have the adequate reference range for each assay to determine whether PTH concentrations are elevated.

Measurement of PTH and Calcium

Question 1A: Do third-generation PTH assays perform better clinically than second-generation PTH assays for the diagnosis of PHPT? The previous publication by the Third International Workshop on Hyperparathyroidism (the last workshop) acknowledged that there are several challenges with the use of the first-generation PTH assay, including the difficulty in discriminating between normal individuals and patients with PHPT or nonparathyroid hypercalcemia.

Question 1B: Do we now have optimal reference intervals for serum PTH, and are these intervals based on individuals who are vitamin D replete? The previous publication from the last workshop acknowledged that we do not have optimal reference intervals for PTH values based on coexisting 25(OH)D concentrations (1). We described the reference intervals that defined repletion based on 25(OH)D of more than 50 nmol/L (20 ng/mL). However, there is controversy over


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Diagnosis of Primary Hyperparathyroidism


Table 1. Sensitivity of Second- and Third-Generation PTH Assays for the Diagnosis of PHPT as Published in the Proceedings of the Third International Workshop (1) Demographic and Biochemical Data

PTH Assay Sensitivity

Mean Cat, mg/dL

Control Group

EDTA plasma

?

60 ⫾ 2

Serum

10.9 ⫾ 0.1

0/39

63.9 ⫾ 8.2

Serum

11.0 ⫾ 0.88

145

28/117

61.7 ⫾ 3.2

Serum

11.2 (9.52– 11.24)

405

30/205

Mean, ⬎60

No Assay range No Assay range Yes 70 Postmenopausal women Yes 74 subjects 25(OH)D ⬎50 nmol/L Variable

No. of Patients

Sex, n (M/W)

Age, y

Samples

Gao, 2001 (3) Silverberg, 2003 (4) Carnevale, 2004 (5) Boudou, 2005 (6)

165

?

?

56

7/49

39

All

Mean, near 11

this threshold. The debate was sharpened with the Institute of Medicine (IOM) using this threshold and The Endocrine Society proposing a higher threshold of 75 nmol/L (30 ng/mL) (7, 8). The thresholds do matter; a meta-analysis of trials of vitamin D supplementation has reported a linear relationship between PTH and 25(OH)D such that a lower reference interval would be obtained if the higher threshold for repletion was used (9). A paradox that has not been resolved is the relationship between circulating 25(OH)D and PTH concentrations when the active metabolite, 1,25(OH)2D, is believed to be the physiological regulator of PTH secretion. It is possible that it is circulating 25(OH)D and not 1,25(OH)2D that influences PTH secretion (10). The use of a vitamin D-replete population to establish a reference range for PTH has been further emphasized by Fillée et al (11) who showed that the upper limit of the PTH reference interval is lower in vitamin D-replete individuals [eg, 25(OH)D ⬎ 50 nmol/L]. In addition, diagnostic accuracy (sensitivity and specificity) for PHPT is also improved in a vitamin D-replete population (11). In the latter study, about 12% of subjects with PHPT had PTH concentrations that were within the normal reference range. A larger case series found 8% of subjects with PHPT whose PTH concentrations were within the normal reference range (12). Virtually all subjects whose PTH concentrations were in the normal reference range were in the upper half of the reference interval. Only about 1% showed PTH concentrations below 40 pg/mL (12). It has been noted that patients with PHPT and normal PTH concentrations experience more delays before surgery, presumably to allow further diagnostic testing (12). These individuals may be more likely to be vitamin D replete (13). Subjects with hypercalcemia and a PTH in the upper range of normal are

Nichol’s Allegro Intact PTH

Scantibodies Laboratory Total PTH

Nichol’s Advantage Intact PTH

Scantibodies Laboratory Whole PTH

151/165 (91.5%) 10 – 65 pg/mL

155/165 (93.9%) 7–36 pg/mL

41/56 (73%) 10.65 pg/mL

54/56 (93%) 5–31 pg/mL 32/39 (82%) 10 – 65 pg/mL

195/221 (88.2%)

Nichol’s Advantage Bio-Intact PTH

30/39 (77%) 8 – 44 pg/mL

136/145 (93.8%) 10 – 46 pg/mL

141/145 (97.3%) 11– 60 pg/mL

122/145 (84.2%) 8.4 –34 pg/mL

1129/145 (89%) 9 – 41 pg/mL

168/184 (91.3%)

141/145 (97.3%)

351/405 (89.1%)

129/145 (89%)

physiologically not normal. It is important to note that such “nonsuppressed” concentrations are entirely compatible with the diagnosis of PHPT. Further studies are required to establish reference intervals for second- and third-generation PTH assays using large population cohorts that are comprised of vitamin D-replete subjects. It would also be of interest to stratify subjects according to age, sex, race, glomerular filtration rate, and possibly even body mass index. One of these potential variables, age, has become apparent in that older and less mobile subjects have less suppression of PTH in response to vitamin D repletion (9, 14). Question 1C: Is normocalcemic hyperparathyroidism a part of the diagnostic spectrum of PHPT? NPHPT, a variant of the traditional hypercalcemic presentation of PHPT, is characterized by consistently elevated PTH concentrations with normal total and ionized serum calcium concentration in the absence of secondary causes for elevated PTH concentrations (15). In 2008, the last workshop acknowledged the existence of this PHPT variant but considered there was a need for more research (1). The diagnosis of NPHPT must include normal serum total calcium and ionized calcium on several occasions. Although there are not enough data to determine the timing or frequency of sampling to establish the diagnosis of NPHPT, this panel suggests that an isolated level of PTH above the upper limit of the normal range should be confirmed on at least two further occasions over a period of 3– 6 months. There have been recent reports that between 4 and 10% of patients with PHPT have normal serum calcium but elevated ionized calcium and PTH (16, 17),


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and therefore these patients would not be considered to have NPHPT. It is also important to exclude disorders that are associated with secondary or compensatory elevated PTH with normal calcium concentrations, such as: 1) vitamin D insufficiency [25(OH)D minimal goal level should be 50 nmol/L, but ⬎75 nmol/L is desirable]: patients initially thought to have NPHPT may become hypercalcemic when 25(OH)D concentrations are raised to ⬎75 nmol/L, making a diagnosis of traditional hypercalcemic PHPT that was masked by vitamin D deficiency; 2) chronic kidney disease (estimated glomerular filtration rate GFR [eGFR] should be ⬎60 mL/min): population studies demonstrate that PTH begins to rise with eGFR ⬍ 60 mL/min; 3) drugs known to increase PTH: thiazides, bisphosphonate, denosumab, and lithium. Whereas the first three drugs promote a physiological elevation of PTH concentration, the latter decreases the sensitivity of the parathyroid gland to circulating calcium and may also reduce urinary calcium excretion, ultimately leading to parathyroid hyperplasia (18, 19). Discontinuation of lithium and thiazides, if medically feasible, should be considered in order to make the diagnosis of NPHPT. PTH elevation with bisphosphonate use appears to be attenuated or reversed with chronic use; with denosumab use, PTH concentrations are elevated for approximately 3 months of the 6-month injection interval (20); 4) hypercalciuria; and 5) occult malabsorption syndromes (15). There is an association between the newly described parathyroid incidentaloma (identified during thyroid ultrasonography) and elevated PTH. In about one-third of these patients, PTH will be elevated with normal serum calcium. With neck ultrasound being used more widely, it is possible that NPHPT will be diagnosed more frequently in this manner (21, 22). Parathyroid function appears to be disordered in NPHPT; the PTH response to an oral calcium load is blunted. Furthermore, the condition may proceed to hypercalcemia as part of the disease course, in which case the hypercalcemic form of the disease becomes evident. In one surgical series, improvement in spine and hip bone density in response to parathyroidectomy for NPHPT was not significantly different from PHPT (23). It has been proposed that NPHPT is the first phase of a biphasic disorder that later may become manifest as hypercalcemic PHPT, occasionally being unmasked by the estrogen deficiency of the menopause (15, 24). In a small cohort of 37 patients with NPHPT, 40% developed signs of PHPT over the course of a mean follow-up of 3.1 years, and 19% developed hypercalcemia (25). There have been few population-based studies, and so the prevalence of this disorder is unclear (24, 26), although it may be between 0.4 and 3.1% (26).


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Thus, NPHPT is part of the diagnostic spectrum of PHPT, and we need to ensure a correct diagnosis and to follow up by serum calcium measurement because these patients may develop hypercalcemia.

Measurement of 25(OH)D Question 2: A, Should practitioners measure 25(OH)D in all patients with suspected PHPT? B, How should the reference ranges for different assays be interpreted? C, What represents the threshold for overtreatment? D, Is it useful to measure 1,25(OH)2D in patients with PHPT and under what circumstances? At the time of the last workshop, it was concluded that vitamin D deficiency was common in PHPT and that measurement of 25(OH)D concentrations was recommended routinely. In patients with vitamin D deficiency, it was recommended that they should be made vitamin D replete before surgery and that serum 25(OH)D be maintained above 50 nmol/L (20 ng/mL). The panel also recognized that standardization of the clinical laboratory measurement of 25(OH)D was needed, that participation in external quality control schemes was advised, and that further research was needed into vitamin D repletion, particularly in the context of randomized controlled trials. In the past 5 years, there has been progress in some, but not all areas. Based upon recommendations of the IOM, vitamin D insufficiency is defined by 25(OH)D concentrations ⬍50 nmol/L and vitamin D deficiency by concentrations ⬍25 nmol/L. The prevalence varies by country, with rates of insufficiency of 56% (China [27]), 60% (Israel [28]), 81% (Denmark [29]), and 93% (France [30]) and prevalence of deficiency of about 30% in Denmark (29) and 50% in India (31). These findings contrast with 1,25(OH)2D concentrations that are generally increased in PHPT, with concentrations above the reference range in 24% (32). In PHPT, the most likely cause for abnormally low vitamin D concentrations is the increased rate of metabolic clearance (24-hydroxylation) induced by 1,25(OH)2D (and possibly PTH) (29). This finding is consistent with the observation that 25(OH)D returns to concentrations found in the normal population after successful parathyroidectomy (33, 34). Other causes to account for low 25(OH)D concentrations in PHPT have been considered, such as excessive weight, but it is not clear that subjects with PHPT are more overweight than subjects without PHPT. Moreover, weight does not decline after parathyroid surgery, yet low 25(OH)D resolves. It has been proposed, but not documented, that there is reverse causality


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in that low 25(OH)D is a causative factor in the development of PHPT (27). If this were likely, it doesn’t explain why abnormally low vitamin D concentrations resolve after successful parathyroid surgery. There are consequences of low 25(OH)D concentrations in PHPT. Low concentrations of 25(OH)D are associated with larger parathyroid adenoma size (35, 36), and the 99mTc-sestamibi scan is more likely to show an abnormal parathyroid gland than in vitamin D-replete subjects (35). Low concentrations of 25(OH)D are associated with higher concentrations of PTH and bone turnover markers. It may take 6 to 12 months for PTH to reduce with repletion. Total alkaline phosphatase activity is higher (28, 32, 35), and concentrations of 1,25(OH)2D are higher along with lower concentrations of phosphate. The high incidence of vitamin D deficiency in India is strongly associated with high alkaline phosphatase activity, and this is thought to contribute to the high prevalence of osteitis fibrosa cystica and brown tumors (58% of cases; Ref. 31). Low 25(OH)D is also related to bone mineral density (BMD) and bone structure. Thus, in a study of BMD in PHPT, a low 25(OH)D level was associated with lower BMD at the femoral neck, distal radius, and whole body, but not the lumbar spine. In a case series of bone biopsies, low 25(OH)D was associated with thinner cortices, although the trabecular bone showed greater volume due to greater trabecular number (37). Tissues other than bone may be affected by low 25(OH)D in PHPT. For example, low 25(OH)D was associated with greater left ventricular hypertrophy (38). Consequences of high concentrations of 1,25(OH)2D have also been demonstrated with higher urinary calcium excretion and lower BMD (32). What information is available on the reversibility of the effects of low 25(OH)D concentrations? In fact, there have been no randomized controlled trials of vitamin D reple-

Table 2.


tion in PHPT. However, in audits of clinical practice and case series, vitamin D repletion has been associated with reduction in PTH concentrations but little effect on mean serum calcium (36, 39, 40). With vitamin D repletion, urinary calcium excretion has been shown to increase in some (40) but not all studies (39). Consequences of excessive repletion of vitamin D in PHPT are not documented. If one uses the IOM value of ⬎125 nmol/L as excessive and associated with vitamin D toxicity, there are three publications that reported such concentrations in PHPT (36, 39, 40). These reports, however, do not provide any specific information about the number of subjects or the consequences of such high concentrations. Conclusions: We recommend that 25(OH)D be measured in all subjects with PHPT. We recommend the cutpoints proposed by the IOM of 50 nmol/L for insufficiency and 25 nmol/L for deficiency, but we also recognize that concentrations ⬎75 nmol/L could be associated with further reductions in PTH concentrations. Cautious replenishment with vitamin D would seem to be prudent in PHPT. No additional information is gained by measuring 1,25(OH)2D, so it is not recommended.

Genetics of Hypercalcemia and PHPT Question 3A: What is the genetic basis for syndromic and nonsyndromic forms of PHPT? More than 10% of patients with PHPT will have a mutation in one of 11 genes (Table 2). Testing for mutations in these genes, which is routinely available, will help to confirm the diagnosis of a syndromic or nonsyndromic form of PHPT, and thereby help in the clinical management and treatment of PHPT patients and their relatives.

Genetic Disorders Associated With PHPT and FHH

Disordera

Gene/Protein

Chromosomal Location

MEN1 MEN2 and MEN3 MEN4 HPT-JT FIHPT

Menin RET CDKN1B (p27, KIP1) CDC73 (Parafibromin) Menin, parafibromin, CASR, CDKN1A (p21), CDKN2B (p15) CDKN2C (p18) CASR CASR GNA11 AP2S1 PTH

11q13 10q11.2 12p13 1q31.2 11q13, 1q31.2, 3q21.1, 6p21.2, 9p21, 1p32 3q21.1 3q21.1 19p13 19q13.2-q13.3 11p15.3-p15.1

NSPHPT FHH1 FHH2 FHH3 nsPHPTb

Abbreviations: NSPHPT, neonatal severe PHPT; nsPHPT, nonsyndromic PHPT. a

Inheritance of all disorders is autosomal dominant, but NSHPT can be recessive.

b

A nonsense PTH has been reported in one patient with PHPT.


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Syndromic and nonsyndromic forms of PHPT may occur as hereditary (ie, familial) disorders, or they may occur as nonfamilial (ie, sporadic) diseases (41, 42). However, this distinction between sporadic and familial forms in PHPT patients may sometimes be difficult because: 1) in some sporadic cases, a family history may be absent because the parent with the disease may not have been investigated or may have died before PHPT symptoms developed; or 2) the PHPT may be due to a de novo germline mutation in the patient, which would account for an absent family history but result in an increased risk of hereditary PHPT in the children of the patient. Syndromic forms of PHPT may occur as part of complex disorders, eg, as part of multiple endocrine neoplasia (MEN) syndromes, and be inherited as autosomal dominant traits (Table 2) (41). Such syndromic PHPT forms associated with complex syndromes comprise MEN syndrome types 1 to 4 (MEN1 to MEN4) and the hyperparathyroidism-jaw tumor (HPT-JT) syndrome (43). As a nonsyndromic isolated endocrinopathy, PHPT may occur as a familial disorder, which is referred to as familial isolated hyperparathyroidism (FIHPT), or as a nonfamilial (sporadic) disorder (44, 45). FIHPT may be due to heterozygous germline mutations of the MEN1, HRPT2 (CDC73), or calcium-sensing receptor (CASR) genes (46 – 49). However, CASR mutations, which lead to loss-of-function of this G protein-coupled receptor, more commonly result in familial hypocalciuric hypercalcemia (FHH) type 1 or neonatal severe primary hyperparathyroidism, which may also be due to homozygous or compound heterozygous CASR mutations (48 –50). FHH1, which is an autosomal dominant disorder, is characterized by lifelong elevations of serum calcium concentrations with low urinary calcium excretion (urinary calcium:creatinine clearance ratio [UCCR], typically ⬍0.01) and normal circulating PTH concentrations in 80% of patients (51, 52). FHH2 and FHH3, which are also autosomal dominant disorders with clinical features that are indistinguishable from those of FHH1, are due to loss-of-function mutations of GNA11, which encodes the G-␣11 subunit (G␣11), and AP2S1, which encodes the adaptorprotein 2 ␴-subunit, respectively (53, 54). Patients with nonfamilial (sporadic) PHPT may also have mutations involving genes that give rise to the hereditary forms of syndromic and nonsyndromic PHPT (Table 1). Thus, approximately 10% of patients presenting with nonfamilial (sporadic) PHPT before the age of 45 years may have a germline MEN1, CDC73, or CASR mutation (55), and this has implications for their future management, in requiring screening for the occurrence of tumors associated with the specific syndrome as well as screening for their children who may inherit the germline


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mutation. Some patients with sporadic PHPT in the sixth to ninth decades have germline mutations of genes encoding cyclin-dependent kinase (CDK) inhibitors (Table 2) (56). Rarely, coexistence of FHH1 and PHPT may be found in a small number of PHPT patients, and it is interesting to note that despite having a CASR mutation, parathyroidectomy in these patients resulted in a reduction of the hypercalcemia or normalization of the serum calcium concentrations (47, 49, 51). Question 3B: What is the value of genetic testing in clinical practice? Genetic testing for mutations is helpful in clinical practice in several ways, including: 1) confirmation of the clinical diagnosis so that appropriate screening for associated tumors can be undertaken; 2) implementation of appropriate treatment, eg, early parathyroidectomy for patients with the HPT-JT syndrome because of the increased occurrence of parathyroid carcinomas, avoidance of minimal invasive parathyroid surgery in MEN1 patients who generally have multigland disease requiring open neck exploration, early prophylactic thyroidectomy in MEN2/ MEN3 patients, and not undertaking surgery in FHH patients; 3) identification of family members who may be asymptomatic but harbor the mutation and therefore require screening for tumor detection and early/appropriate treatment; and 4) identification of the 50% of family members who do not harbor the familial germline mutation and can therefore be reassured and alleviated of the anxiety burden of developing future tumors (41, 42). This latter aspect cannot be overemphasized because it helps to reduce the cost to the individuals and their children, and also to the health services in not having to undertake unnecessary biochemical and radiological investigations. Question 3C: What should be the clinical approach to gene testing in a patient with hypercalcemia? In a patient with hypercalcemia and either high PTH or PTH in the upper half of the reference interval, the key differential diagnosis is between FHH and PHPT (Figure 2). If there are features in the history that signify syndromic PHPT, then the algorithm in Figure 3 is followed. Otherwise, it is critical to measure the UCCR ratio. Patients with FHH have been misdiagnosed to have PHPT because 20% have elevated plasma PTH concentrations (51). In addition, 20% of FHH patients may have a UCCR ⬎0.01 (51, 52), and therefore be indistinguishable from patients with PHPT; moreover, low UCCRs are observed in PHPT patients with vitamin D deficiency, or renal insufficiency, or African-American origins (57, 58). These findings indicate the limitations of using elevated plasma PTH concentrations and UCCRs to discriminate between


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Patient with hypercalcemia (plasma Ca++ up to 3.0mmol/L) and normal or high plasma PTH; not taking drugs (e.g. thiazide or lithium) or vitamin D preparations

Assess for family history of PHPT and for syndromic forms of PHPT

NO

YES

Measure: UCCR Serum 25 (OH) D eGFR

Proceed to genetic testing – as described in Figure 3

UCCR >0.02

UCCR = 0.01 to 0.02 25(OH)D >50nmol/L eGFR >60ml/min

UCCR 50nmol/L eGFR >60ML/min

PHPT (sporadic form) >90% likelihood. Proceed to figure 3

Results are not able to distinguish between PHPT and FHH

FHH >95% likelihood

Proceed to gene testing of CASR, GNA11 and AP2S1 genes, which may confirm FHH1, FHH2 and FHH3, respectively

Consider gene testing of CASR, AP2S1 and GNA11 to confirm diagnosis of FHH, and to facilitate screening / diagnosis in relatives

Figure 2. Clinical approach to distinguishing between PHPT and FHH in a hypercalcemic patient. Less than 5% of patients presenting with nonfamilial (sporadic) and nonsyndromic PHPT, due to solitary parathyroid adenomas in the sixth to ninth decades of life, may have rare variants/ mutations of CDKN1A, CDKN2B, or CDKN2C. Urinary calcium excretion is highly variable and should be estimated on at least two or three occasions on different days. In addition, urinary calcium excretion may be low in patients of African-American origin or those with vitamin D deficiency or renal impairment (18 –21). Ca⫹⫹, calcium; AP2S1, adaptor protein 2 ␴-subunit; GNA11, G protein ␣11 subunit.

PHPT and FHH and favor the use of mutational analysis of the CASR, GNA11, and AP2S1 genes to identify patients with FHH1, FHH2, and FHH3, respectively (48 – 50, 53, 54), particularly in patients with UCCR between 0.01 and 0.02 (Figure 2).

For patients in whom PHPT is the most likely diagnosis, genetic testing is indicated in some situations (Figure 3). The identification of a germline mutation should prompt entry into appropriate periodic clinical, biochemical, and radiological screening programs, eg, for MEN- and HPT-


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Patient with PHPT

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a

b

YES

Young age , multigland disease (>2 glands), c parathyroid carcinoma, or atypical adenoma st Obtain family history to ascertain if 1 degree family relatives are affected, and if appropriate, d evidence of hypercalcemia in these relatives .

NO Also assess clinically for MEN syndrome, HPT-JT, FHH

NO Sporadic PHPT, e No genetic testing

YES

Relative(s) affected with hypercalcaemia but have no abnormalities of MEN, HPT-JT or FHH, consistent with FIHP Mutational analysis (in order of likely frequency): 1. MEN1 f 2. CASR, AP2S1, GNA11 f 3. HRPT2 (CDC73) e, f 4. CDKN -1A, -1B, -2B, -2C f 5. RET 6. PTH

Mutation detected. Follow up by regular screening for the development of appropriate tumors for the MEN syndromes or HPT-JT

Patient or relative has abnormalities, consistent with either an MEN syndrome, HPT-JT or FHH. Pursue appropriate investigations including gene analysis (e.g. MEN1, CDC73, CASR, AP2S1, GNA11)

Mutation or abnormality not detected, indicating that likelihood of a MEN syndrome, HPT-JT or FHH is low

Assess first-degree relatives Figure 3. Clinical approach to genetic testing in a patient with PHPT. AP2S1, adaptor protein 2 ␴-subunit; GNA11, G protein ␣11 subunit; HRPT2, hyperparathyroidism type 2; CDC73, cell division cycle 73; CDKN, cyclin-dependent kinase inhibitor; RET, rearranged during transfection proto-oncogene. lsqb]Reproduced from R. V. Thakker: Familial and hereditary forms of primary hyperparathyroidism. In: Bilezikian JP, Marcus R, Levine M, Marcocci C, Potts JT, Silverberg S, eds. The Parathyroids: Basic and Clinical Concepts, with permission. 3rd ed. Atlanta, GA: Elsevier (in press).]

JT-associated tumors (41, 42). The absence of clinical manifestations of hereditary or syndromic forms of PHPT and any genetic abnormalities within the 11 genes would indicate that the likelihood of a MEN syndrome, HPT-JT

syndrome, or FHH was low (ie, ⬍5%) (41, 42). Firstdegree relatives of a PHPT patient with a mutation should be identified and offered genetic counseling and appropriate gene testing, and individuals who have inherited the


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mutation should be offered periodic screening, even if they are asymptomatic. First-degree relatives who have not inherited the causative mutation require no further follow-up and may be alleviated of the anxiety associated with the development of MEN- or HPT-JT-associated tumors. Genetic testing should use DNA obtained from leukocytes, salivary cells, skin cells, or hair follicles (ie, nontumor cells) because DNA from parathyroid tumors is not clinically useful for establishing the diagnosis or staging, as such tumors may contain multiple mutations. For example, two studies that have analyzed the whole-exome sequences of parathyroid adenomas from nonfamilial (sporadic) PHPT patients have reported that the number of somatic (ie, tumor-specific) mutations in a parathyroid tumor may range from two to 110, and that between 35 and 50% of such parathyroid tumors will have a somatic mutation involving the MEN1 gene (59, 60). It is important to emphasize that best clinical practice for such genetic testing should include agreement (ie, informed consent) from the patient and access to genetic counselors (41). Genetic testing should be performed by accredited centers, some of which can be contacted using the following links: http://www.ncbi.nlm.nih.gov/sites/ GeneTests/ (giving details of centers in Canada, Denmark, Greece, Israel, Japan, and the United States); http://www. orpha.net/consor/cgi-bin/index.php or www.eddnal.com (giving details of centers in Austria, Belgium, Denmark, Finland, France, Germany, Holland, Ireland, Italy, Norway, Portugal, Spain, Sweden, Switzerland, and the United Kingdom). The price currently charged (April 2014) in the United Kingdom for the genetic tests included in Figure 3 is US $2100. This figure is likely to decrease as technology improves. We conclude that a genetic test is indicated in some patients with PHPT and hypercalcemia who are at high risk of carrying a mutation.

Acknowledgments Address all correspondence and requests for reprints to: Professor Richard Eastell, Metabolic Bone Centre, Northern General Hospital, Herries Road, Sheffield S5 7AU, United Kingdom. E-mail: [email protected]. Disclosure Summary: R.E. provides consulting advice to Roche Diagnostics and Immunodiagnostics Systems. All other authors have nothing to disclose.

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