Calcium-Sensing Receptor (CASR) Mutations in Hypercalcemic States: Studies from a Single Endocrine Clinic Over Three Years

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1 ORIGINAL ARTICLE Endocrine Care Calcium-Sensing Receptor (CASR) Mutations in Hypercalcemic States: Studies from a Single Endocrine Clinic Over Three Years Vito Guarnieri,* Lucie Canaff,* Francisco H. J. Yun,* Alfredo Scillitani, Claudia Battista, Lucia A. Muscarella, Betty Y. L. Wong, Angelantonio Notarangelo, Leonardo D Agruma, Michele Sacco, David E. C. Cole, and Geoffrey N. Hendy Departments of Laboratory Medicine and Pathobiology, Medicine, and Genetics (F.H.J.Y., B.Y.L.W., D.E.C.C.), University of Toronto, Ontario, Canada M5G 1L5; Genetics (V.G., L.A.M., A.N., L.D A.), Endocrinology (A.S., C.B.) and Pediatrics (M.S.) Units, Hospital Casa Sollievo della Sofferenza, Istituto di Ricovero e Cura a Carrattere Scientifico, San Giovanni Rotondo (Foggia), Italy; Departments of Medicine, Physiology, and Human Genetics (L.C., G.N.H.), McGill University; and Calcium Research Laboratory, and Hormones and Cancer Research Unit, Royal Victoria Hospital, Montreal, Quebec, Canada H3A 1A1 Context: Inactivating mutations of the calcium-sensing receptor (CASR) are implicated in different hypercalcemic syndromes, including familial hypocalciuric hypercalcemia (FHH), primary hyperparathyroidism (PHPT), and familial isolated hyperparathyroidism (FIHP). However, molecular diagnostics applied to large nonselected hypercalcemic cohorts from a single center have not been reported. Objective: Our objective was to describe the prevalence, type, and potential pathogenicity of CASR mutations in a series of cases with FHH (n 17), PHPT (n 165), and FIHP (n 3) and controls (n 198) presenting at a single endocrine clinic. Subjects: All were prospectively evaluated at the Casa Sollievo della Sofferenza Hospital in southern Italy over a 3-yr period. Methods: CASR screening was conducted by denaturing HPLC. The variant CASRs were functionally characterized by transient transfection studies in kidney cells in vitro. Results: A single novel missense variant was identified in one PHPT case. However, in FHH probands, mutations were found in eight of 17 (47%). With a hypercalcemic family member, mutation detection rate in FHH rose to seven of eight (87%), whereas only one of nine sporadic cases was positive, and none of the three FIHP cases had detectable CASR mutations. Five missense variant CASRs, identified in control subjects, performed as wild type in functional assays, whereas the missense mutant CASRs identified in the FHH patients, and in the one PHPT case, exhibited significant impairment. A novel intronic mutation (IVS4-19a3c) found in one FHH family, created an abnormally spliced product in an in vitro minigene assay. Conclusion: CASR testing, with functional analysis, provides critical confirmatory evidence in the differential diagnosis of hypercalcemic states. (J Clin Endocrinol Metab 95: , 2010) ISSN Print X ISSN Online Printed in U.S.A. Copyright 2010 by The Endocrine Society doi: /jc Received November 7, Accepted January 14, First Published Online February 17, 2010 * V.G., L.C., and F.H.J.Y. contributed equally to this study. Abbreviations: CASR, Calcium-sensing receptor; DHPLC, denaturing HPLC; ECD, extracellular domain; FHH, familial hypocalciuric hypercalcemia; FIHP, familial isolated hyperparathyroidism; HPT-JT, hyperparathyroidism-jaw tumor syndrome; MEN, multiple endocrine neoplasia; NSHPT, neonatal severe hyperparathyroidism; PHPT, primary hyperparathyroidism. J Clin Endocrinol Metab, April 2010, 95(4): jcem.endojournals.org 1819

2 1820 Guarnieri et al. CASR Mutations in a Hypercalcemic Cohort J Clin Endocrinol Metab, April 2010, 95(4): The calcium-sensing receptor (CASR) is a plasma membrane G protein-coupled receptor abundantly expressed in the parathyroid gland and kidney tubule where it plays a key role as the calciostat in maintaining extracellular fluid calcium concentrations within a narrow normal range (1). Activation of CASR by increased ionized calcium leads to decreased PTH secretion and increased calcium excretion. In several hypercalcemic disease states, CASR function may be impaired by inactivating mutations in the gene or reduced levels of expression (2). The human CASR (MIM ) is encoded by six exons (exons II VII) of the gene (3 6) located on chromosome 3q13.3-q21 (7). Exons IA and IB encode alternative 5 -untranslated regions (8 10), exon II encodes the common 5 -untranslated region, the ATG initiation codon, the signal peptide sequence, and the beginning of the extracellular domain (ECD); exons III VI encode the main part of the ECD, and exon VII encodes the end of the ECD, the transmembrane domain, and the intracellular domain. The human receptor consists of 1078 amino acids, with approximately 600 amino acids in the ECD, 250 in the transmembrane domain, and 216 in the cytoplasmic tail. In most instances, heterozygous inactivating mutations of the CASR gene cause familial hypocalciuric hypercalcemia (FHH, MIM ), whereas homozygosity manifests as neonatal severe hyperparathyroidism (NSHPT, MIM ) (11). The hypercalcemia of FHH is mild to moderate, similar to that of mild primary hyperparathyroidism (12, 13). Blood ionized calcium is usually within 10% of the upper limit of normal, although occasional families exhibit higher calcium concentrations. In the majority, calcium excretion is low relative to the hypercalcemia. The calcium-to-creatinine clearance ratio is generally less than 0.01 in FHH, whereas it is higher in primary hyperparathyroidism and other hypercalcemic disorders. Children with NSHPT may develop severe, symptomatic hypercalcemia with skeletal changes of unremitting hyperparathyroidism (11). Delay in effective treatment (total parathyroidectomy) can lead to devastating neurodevelopmental damage, if it is not fatal (14). However, some de novo mutations of the CASR gene present with milder, less severe neonatal hyperparathyroidism disease that can be transient or self-limiting. Over 150 inactivating mutations in the CASR have been described in association with FHH and/or NSHPT (15). The majority are missense, with others being nonsense, insertion, and deletion/insertion (16). Primary hyperparathyroidism (PHPT) is a common cause of hypercalcemia in adults, and the incidence has been estimated to be as high as 1 in 1000 (2). The important laboratory features are elevated serum calcium, decreased fasting serum phosphate (albeit commonly within the normal range), increased serum PTH, and normal or frankly increased urinary calcium excretion. The sporadic disorder occurs most frequently in the sixth decade, but it is uncommon in children and is more likely then to be a component of a familial endocrinopathy. Somatic CASR mutations are not a significant factor in parathyroid tumorigenesis (17). Ten percent of PHPT cases are hereditary and can occur as an isolated condition or as part of a syndrome. These syndromes are FHH/NSHPT, multiple endocrine neoplasia (MEN) type 1 (MEN1) and type 2 (MEN2), the hyperparathyroidism-jaw tumor syndrome (HPT-JT), and familial isolated hyperparathyroidism (FIHP) (2). Mutations of the CASR have been identified in a few kindreds with FIHP (18 20). Various polymorphic variants have been identified in the CASR gene (21). Of note, three single-nucleotide polymorphisms (SNPs) in exon VII encode nonconservative amino acid changes (A986S, R990G, and Q1011E) in the COOH-terminal tail of the CASR. These polymorphisms are predictive of serum calcium concentrations in some normal Caucasian populations, either individually or in haplotype combination (22 24). In PHPT patients, there is a significant association with the SRQ haplotype, suggesting it as a susceptibility marker for the development of PHPT. PHPT patients having the AGQ haplotype are at greater risk of developing renal stones (25). Identification of CASR mutations plays an important role in the clinical management of inherited hypercalcemic disorders. In the present study, we report on a comprehensive search for CASR mutations across all the various hypercalcemic cases seen over a 3-yr time period in a single endocrine clinic. Subjects and Methods Patients Patients for this study were ascertained at the Endocrine Clinic specializing in bone and mineral disorders at the San Giovanni Rotondo Centre (Istituto di Ricovero e Cura a carattere Scientifico, Casa Sollievo della Sofferenza, Italy) over a 3-yr period ( ). Serum ionized calcium, serum total calcium (albumin-adjusted), phosphate, creatinine, PTH, and calcium excretion (as calcium to creatinine clearance ratio) were measured by routine laboratory techniques. Medical histories were reviewed to confirm that phenotypic features were consistent with one of three clinical conditions: sporadic PHPT, FHH, or FIHP. From an initial pool of cases, 165 met the criteria for inclusion as PHPT, namely, elevated serum calcium, inappropriately high serum PTH, calcium excretion higher than 0.01, and absence of hypercalcemic relatives (see Table 1). In the PHPT group, there were 29malesand136females[age56 14yr(mean SEM)].Abouttwo thirds of these (107 of 165) underwent parathyroid surgery (Table 1). By histopathology, adenomas were present in about 80% (83 of 107), and the majority of the rest had hyperplasia. Normal parathyroid glands were identified in five cases (Table 1).

3 J Clin Endocrinol Metab, April 2010, 95(4): jcem.endojournals.org 1821 TABLE 1. Clinical characteristics of study subjects Controls (n 198) PHPT (n 165) FHH (n 17) FIHP (n 3) Serum calcium alb-adj (mg/dl) a c b b,c Serum ionized calcium (mmol/liter) a c b b,c Serum phosphate (mg/dl) a b a b Serum creatinine (mg/dl) a b a,b a,b Serum PTH (pg/ml) a b a b Urinary calcium to creatinine clearance ratio ND a b a Parathyroid gland histopathology NA Adenoma 83 2 d 1 Hyperplasia Normal 5 e 1 0 NA, Not applicable; ND, not determined. a c Pairwise mean values followed by different superscript letters are significantly different from one another, by one-way ANOVA and Tukey post hoc testing. d One subject, considered FHH because of documented hypocalciuria, was noted subsequently to have worsening hypercalcemia and hyperparathyroidism and underwent surgery whereupon an adenoma was identified. The patient s DNA was then subjected to further genetic analysis, and a MEN1 mutation was identified. The other subject was noted to have increasing serum PTH (247 pg/ml; normal 72 pg/ml), renal stones, and an extrathyroidal nodule. Despite the documented hypocalciuria, parathyroid surgery was performed, and an adenoma was found and removed. e One subject underwent repeat surgery, but no gland was identified, and the patient remains hypercalcemic and hyperparathyroid but only mildly symptomatic.the other four patients declined repeat surgery. Three cases of FIHP were identified (three women, yr), based on the finding of PHPT in at least one individual (the index case) and hypercalcemia with hypercalciuria in at least one first-degree relative (see pedigrees in Fig. 1). To exclude other genetic causes of FIHP, mutation searches of MEN1 and HRPT2 (the HPT-JT gene) were performed and were negative in all three families. The three index cases underwent parathyroidectomy, as did two first-degree relatives. Three (two index cases and one relative) had adenomas and two (one index case and one relative) had parathyroid hyperplasia. Seventeen patients were classified as FHH if they or a family member had elevated serum calcium, inappropriately normal/ high serum PTH, and relatively low calcium to creatinine clearance ratio ( 0.01) by at least two independent urine samples in the absence of any medication affecting renal calcium clearance (e.g. thiazides). Pedigrees for these 17 subjects (four males and 13 females, yr) are presented in Fig. 1. Note that both familial and sporadic hypocalciuric hypercalcemic individuals are categorized under the term FHH. Control subjects One hundred ninety eight control subjects were recruited from the local blood donor clinic after exclusions for identical surnames and those individuals on medication or with comorbidities influencing bone or mineral metabolism. Mean age of this group was yr (104 males and 94 females). Serum ionized calcium, serum total calcium (albumin adjusted), phosphate, creatinine, and PTH levels were measured and all were normal. DNA amplification, denaturing HPLC (DHPLC), and DNA sequencing Genomic DNA was extracted from peripheral white blood cells using standard methods. The CASR protein-coding regions and intron/exon boundaries were PCR amplified as 13 fragments ranging in size from bp. Twelve amplicons were subjected to DHPLC (26). Any fragment (from an experimental sample) that generated a modified DHPLC melting curve compared with the control (from wild type) was further examined by DNA sequencing (26). The 13th and most 3 fragment, harboring the three common polymorphisms, A986S, R990G, and Q1011E, encoded by exon VII, was analyzed immediately by DNA sequencing rather than prescreened by DHPLC. Functional study and bioinformatics Other methods including in vitro functional and bioinformatics analysis of all mutations identified are in the Supplemental Methods (published on The Endocrine Society s Journals Online web site at Databases The web sites for data in this article are as follows: Online Mendelian Inheritance in Man, omim/ (CASR #601199, familial hypocalciuric hypercalcemia #145980, neonatai severe hyperparathyroidism #239200, familial isolated hyperparathyroidism #145000); CASR GenBank X81086; calcium-sensing receptor database mcgill.ca; and Genetests All subjects All subjects gave informed consent for the study that was approved by the ethics committee at the Casa Sollievo della Sofferenza Hospital. Controls and PHPT patients were also subjects in an earlier study restricted to an analysis of common CASR polymorphisms (25). Results Patient characteristics Summary statistics (one-way ANOVA) for the clinical characteristics of the four groups are in Table 1.

4 1822 Guarnieri et al. CASR Mutations in a Hypercalcemic Cohort J Clin Endocrinol Metab, April 2010, 95(4): FIG. 1. Pedigrees of families with PHPT (A), FIHP (A C), and FHH (A G, I, J, K, L, and N) and FHH individuals (H, M, O, P, and Q). For each, either the particular heterozygous CASR mutation found or the fact that no mutation was identified (NI) is indicated. For individuals within a pedigree, CASR genotype status is indicated by white symbols (wild type or status unknown) and black symbols (mutant). Probands are indicated by an arrow: the biochemical details of each tested individual are given below their symbol. Reference intervals are as follows: blood ionized calcium (ica), mmol/liter; albumin-adjusted serum calcium (Ca adj ), mg/dl; serum phosphate (P), mg/dl; total serum alkaline phosphatase activity (ALP), IU/liter; serum PTH, pg/ml; serum 25-hydroxyvitamin D (25OH-D), ng/ml; urine calcium/creatinine in milligrams per deciliter (Ca/ Cr), less than 0.2. The vitamin D status may influence the presentation of both FHH and PHPT (42), thus increasing the difficulty in differentiating the two conditions. To provide reassurance that categorization of the sporadic hypocalciuric hypercalcemia cases as FHH individuals (see above) was appropriate, a reevaluation of the biochemical parameters was made in those FHH cases (both family probands and sporadic individuals) in which vitamin D deficiency was noted initially, after they had been vitamin D repleted and their serum 25-OHD levels brought to the sufficient or near-sufficient range. All cases remained hypocalciuric supporting the FHH classification (see Supplemental Table 1). Subjects without an identified CASR mutation that had low urine Ca/Cr but as probands have not yet been shown to transmit this to the next generation therefore can be said to have hypocalciuric hypercalcemia of unknown etiology. Means for serum total calcium (after albumin adjustment) and ionized calcium were higher in all three patient groups compared with controls, but levels were higher still in the PHPT and FIHP groups compared with FHH. Serum phosphate in the FHH group was not different from controls but was significantly lower in PHPT and FIHP. Serum creatinine was significantly higher in the PHPT group only, as was the degree of hypercalciuria. Although the majority of the FHH group had hypocalciuria, borderline calcium clearance ( but 0.020) was not uncommon (Fig. 1) in either index cases or hypercalcemic relatives. CASR variants in control and patient groups The identified sequence variants are categorized according to subject group (Table 2). Control subjects Sequence variants were detected in 4% (eight of 200) of the controls: missense (n 5; Y20H, P274S, R285W, E755D, and C851S) and synonymous (n 3; F688F, E870E, and A988A). FHH patients Sequence variants were detected in 53% (nine of 17) of FHH subjects: missense [n 5; P55L, T263M, F351V (in

5 J Clin Endocrinol Metab, April 2010, 95(4): jcem.endojournals.org 1823 TABLE 2. CASR sequence variants identified in this study Subject category No. of subjects Type Nucleotide change Trivial name Amplicon Protein domain C 1 Missense c.60 C3 T Y20H E2-2 ECD Novel C 1 Missense c.820 C3 T P274S E4-1;E4-2 ECD Novel C 1 Missense c.853 C3 T R285W E4-2 ECD Novel C 1 Missense c.2265 G3 T E755D E7-3 ECL Novel C 1 Missense c.2551 T3 A C851S E7-4 TM7 Baron et al. (27) C 1 Silent c.2064 C3 T F688F E7-2 TM3 Novel C 1 Silent c.2610 G3 A E870E E7-4 ICD Yun et al. (21) C 1 Silent c.2964 C3 G A988A E7-5 ICD Novel FHH 1 Missense c.164 C3 T P55L E2-2 ECD Heath et al. (6) FHH 1 Missense c.788 C3 T T263M E4-1;E4-2 ECD Novel FHH 2 Missense c.1051 T3 G F351V E4-2 ECD Novel FHH 1 Missense c.1393 C3 T R465W E5 ECD Novel FHH 1 Missense c.3235 T3 C X1079QextX1087 E7-5 ICD Novel FHH 1 Frameshift c.2383delc R795GfsX836 E7-3 ICL Nissen et al. (28) FHH 1 Silent c.2244 C3 G P748P E7-3 ECL D Souza-Li et al. (37) FHH 1 Intronic c A3 C IVS4-19 E5 Yun et al. (21) FHH 1 Silent c.2610 G3 A E870E E7-4 ICD Yun et al. (21) PHPT 1 Missense c.848 T3 C I283T E4-2 ECD Novel PHPT 1 Silent c.573 G3 A E191E E4-1 ECD Yun et al. (21) PHPT 2 Silent c.2610 G3 A E870E E7-4 ICD Yun et al. (21) C, Control; ECD, extracellular domain; ECL, extracellular loop; ICD, intracellular domain; ICL, intracellular loop; TM, transmembrane. Ref. two cases), R465W, and X1079QextX1087], synonymous (n 2; P748P and E870E), frameshift (n 1; R795GfsX836), and intronic (n 1; c a3c). The T263M and E870E variants were both found in a single sporadic case, but phase, i.e. whether the two variants are together on one of the two homologous chromosomes, could not be established. The mutation discovery rate in sporadic cases was only one in nine, significantly lower (P 0.003, Fisher exact test) than in FHH kindreds (seven in eight) with the hypocalciuric hypercalcemia phenotype directly ascertained in family members. PHPT patients. Sequence variants were detected in 2.4% (four of 165) of the PHPT patients: missense (n 1; I283T) and synonymous (n 3; E191E in one subject and E870E in two). The novel I283T mutation was identified in a 56-yr-old woman with hypertension, a history of renal stones (20 yr previously) and osteoporosis (bone mineral density T-scores: lumbar spine 3.2 and femoral neck 3.0), but no fractures and a typical PHPT profile (serum Ca 11.9 mg/dl, phosphate 2.5 mg/dl, creatinine 0.76 mg/dl, PTH 143 pg/ml, and urinary calcium clearance ratio 0.025). Ultrasound of the neck revealed a suspected nodule behind the left lobe of the thyroid, and she underwent parathyroidectomy with removal of a typical adenoma. After surgery, her serum calcium was 9.8 mg/dl and her calcium excretion 100 mg/d, but thiazide treatment precluded formal evaluation, and further information was not available. FIHP patients. No variants were identified in three patients with FIHP. Further screening for HRPT2 and MEN1 mutations was also negative (data not shown). Functional characterization of CASR missense variants A functional analysis of CASR variants was made by transient transfection studies in HEK293 cells. The data demonstrate that 1) the variants, Y20H, P274S, R285W, E755D, and C851S, identified in controls, functioned identically to the wild-type CASR with respect to intracellular expression, maturation, cell surface expression, and signaling, and 2) the FHH mutants, T263M, F351V, R465W, X1079QextX1087, and R795GfsX836, and the PHPT mutant, I283T, were impaired relative to the wildtype CASR with respect to maturation, cell surface expression, and signaling (Fig. 2) (see Results, Fig. 2, and Supplemental Table 2). Aberrant expression of the splicing variant in HeLa cells An a3c transversion at position 19 in IVS4 (c ) was identified in the proband of FHH kindred D. No other variant sequence was found. The IVS4 19 variant segregated with the FHH trait being present in the father and his two daughters (all affected) but not in his unaffected son. This variant was not found in our survey of more than 1100 Caucasian control chromosomes or in other ethnic groups (21). To study the consequences of the

6 1824 Guarnieri et al. CASR Mutations in a Hypercalcemic Cohort J Clin Endocrinol Metab, April 2010, 95(4): IVS4 19 mutation genomic fragments comprising CASR exon V and flanking intron sequences representing both the wild-type and mutant alleles of the proband were PCR amplified and cloned into a minigene construct of the exon-trapping type. Constructs were transiently transfected into HeLa cells and RNA extracted, and RT-PCR products were analyzed. Cells transfected with the basic minigene construct demonstrated a processed PCR product of 434 bp (with vector-alone transfected cells being negative) (Fig. 3A) consistent with the proper splicing of exons A, B, and C of the minigene (Fig. 3B). Cells transfected with the minigene having wild-type CASR sequence (MG-CASR-WT) had a spliced transcript PCR product of 665 bp, whereas cells transfected with the minigene having the mutant CASR sequence (MG-CASR-MUT) had some wild-type product (665 bp) and a second product of 547 bp (Fig. 3C). Nucleotide sequence of the PCR products (Fig. 3D) demonstrated that whereas the 665-bp product represented proper splicing of exons A, CASR V, B, and C (Fig. 3E), the 547-bp product represented aberrant splicing in which exon A spliced to a cryptic site within CASR exon V. Otherwise, the splicing was normal (Fig. 3F). In summary, in the mutant transcript, the normal AG acceptor site in IVS4 was ignored, and a cryptic AG site some 118 bp into CASR exon V was used instead (Fig. 3G). FIG. 2. Functional characterization of CASR variants. A, Western blot analysis of cell extracts of HEK293 cells transiently transfected with empty vector, pcdna 3.1 (V), wild-type (WT), or mutant (as indicated) c-myc-tagged CASR cdnas. Upper panel, Recombinant proteins stained with c-myc monoclonal antibody 9E10; lower panel, endogenous proteins stained with a -tubulin antibody. B, CASR cell surface expression detected by ELISA with the c-myc 9E10 mouse monoclonal antibody in HEK293 cells transiently transfected with empty vector, pcdna 3.1 (V), wild-type (WT), or mutant (as indicated) c-myc-tagged CASR cdnas. The absorbance was read at 405 nm. Values shown are the means ( SEM) of three replicate experiments. *, P 0.05 compared with the WT group. C, Extracellular Ca 2 -dependent activation of endogenous ERK1/2 phosphorylation in HEK293 cells transiently transfected with empty vector, pcdna 3.1 (V), wild-type (WT), or mutant (as indicated) c-myc-tagged CASR cdnas. The transfected cells were exposed to 5mM Ca 2 for 5 min, and extracts were made and immunoblotted. Western blots were developed with antibodies against either phospho-erk1/2 (P-ERK1/ 2 or total ERK1/2 (T-ERK1/2). Representative results are shown in the inset. Protein signals were quantitated by densitometry, and the ratios of phospho- ERK1/2 to total ERK1/2 (phospho-erk1/2 accumulation) were determined and normalized to the value obtained in cells transfected with empty vector, pcdna3.1 (V). Values shown are the means ( SEM) of three replicate experiments. *, P 0.05 compared with the WT group. Bioinformatics Predicted effects of all novel amino acid substitutions are summarized in Table 3. None of the programs assessed the stop codon mutation (X1079Q). The prediction algorithms scored Y20H, P274S, and E755D variants in controls as benign, but Polyphen scored R285W as damaging. In contrast, both Polyphen and SIFT scored all four missense mutations found in patients (FHH, n 3; PHPT, n 1) as possibly or probably damaging and not tolerated. However, three of the four mutations, T263M, R465W, I283T, were not predicted by the SNPs3D algorithm to affect structure, although the reliability of the T263M prediction was low. Discussion Findings in control subjects and patient groups Control subjects Sequence variants (apart from the common COOHterminal polymorphisms) were detected in eight control samples. Of the five missense and three synonymous changes, four of the missense mutations were novel but were not considered deleterious by the bioinformatics analysis and were confirmed as such by the in vitro func-

7 J Clin Endocrinol Metab, April 2010, 95(4): jcem.endojournals.org 1825 FIG. 3. Minigene analysis demonstrates aberrant splicing of the CASR IVS4-19a3c transcript. The basic minigene construct (MG) was generated by inserting a cassette consisting of PTHR1 exon I (designated here as exon A), part of PTHR1 IVS1, part of RAB1B IVS4, RAB1B exon V (designated here as exon B), RAB1B IVS5, and RAB1B exon VI (designated here as exon C) into the polylinker of the pcdna3.1d/v5-his-topo mammalian expression vector (V) under the control of the CMV promoter (as described in the Supplemental Methods). MG-CASR-WT and MG-CASR-MUT minigene constructs were prepared by inserting a NotI-XhoI fragment comprising part of CASR IVS4, exon V, and part of IVS5 (representing either CASR wild-type sequence or the CASR IVS4-19a3c mutant sequence) in the intron between exon A and exon B. Constructs were transfected into HeLa cells, and RNA from cell extracts was subjected to RT-PCR using a forward primer in exon A and a reverse primer in exon C. A, Agarose gel electrophoresis of RT-PCR products from cells transfected with either vector (V) alone or the basic minigene (MG) construct. B, Schematic of the basic minigene construct insert showing predicted splicing of exons A, B, and C to yield an RT-PCR product of 434 bp derived from the mrna transcript with the primers used. This was experimentally confirmed by sequence analysis of the product shown in the MG lane of A (data not shown). C, Agarose gel electrophoresis of RT-PCR products of cells transfected with either vector (V) alone or the CASR wild-type (MG-CASR-WT) or mutant (MG-CASR-MUT) minigene constructs. By direct sequencing, the MG-CASR-WT 665-bp product was shown to correspond to a correctly spliced product, and the MG-CASR-MUT 547-bp product was shown to represent an altered splice product lacking the initial 118 bp of CASR exon V. Note that a small amount of the 547-bp product was seen with the MG-CASR-WT construct, indicating the potential for aberrant or leaky splicing of the wild-type pre-mrna. D, Part of the sequence chromatograms of the CASR WT 665-bp product showing normal splicing of exon A to CASR exon V and of the CASR MUT 547-bp product showing abnormal splicing to a site 118 bp into CASR exon V. E, Schematic of the MG- CASR-WT construct insert showing predicted splicing of exons A, CASR V, B, and C to yield an RT-PCR product of 665 bp derived from the mrna transcript as confirmed experimentally by the data of C and D. F, Schematic of the MG-CASR-MUT construct insert showing splicing of exons A, part of CASR V, B, and C to yield an RT-PCR product of 547 bp consistent with the data of C and D. G, The CASR IVS4-19a3c mutation leads to loss of the use of the normal acceptor site at the beginning of exon V. Instead, a cryptic AG acceptor site is used that lies 118 bp inside exon V. The mutation lies in the 20- to 30-bp pyrimidine-rich region typically found in introns between the branch point and the invariant ag acceptor site at the downstream end of CASR IVS4. The deletion of 118 bases of the normal transcript resulting in a frame shift and premature translation termination codon within exon VII (the last exon of the CASR gene) would generate the truncated protein, Q459fsX588. Thus, although the transcript might be predicted to escape nonsense-mediated decay, the resulting protein would be nonfunctional. tional analysis (see below), which is consistent with the normal clinical profile. The fifth missense variant, C851S, was identified previously in members of a family with autosomal dominant hypoparathyroidism (27); the variant did not segregate with hypocalcemic individuals having an activating CASR mutation. Therefore, C851S, as well as Y20H, P274S, R285W, and E755D, are rare, nondeleterious polymorphic variants; with the exception of C851S,

8 1826 Guarnieri et al. CASR Mutations in a Hypercalcemic Cohort J Clin Endocrinol Metab, April 2010, 95(4): TABLE 3. Bioinformatic analysis of novel missense variants Trivial name PHEN score a SIFT score b SNPs3D score c SignalP Segregation with score Conserved d hypercalcemia e Functional analysis Controls Y20H NP Normal C NA Normal P274S NA C NA Normal R285W NA C NA Normal E755D NA V f NA Normal Patients T263M NA V g 0 Impaired F351V NA C Impaired R465W NA V h Impaired X1079Q NP NP NP NA C Impaired I283T NA 0 0 Impaired NA, Not applicable; NP, no prediction. a Score interpretations: 0.5 is benign; 0.5 score 2.0 is possibly damaging; 2.0 score is probably damaging. Scores predicting some deleterious effect are in bold. b Score interpretation: 0.05 is predicted to affect protein function (bold); otherwise tolerated. c Positive score predicts nondeleterious change. Absolute values greater than 0.5 are significantly more accurate predictors. d C,100% conserved in CASR; 0, not conserved; V, conserved in higher vertebrate homologs. e 0, No transmissions;, at least one concordant segregation;, four or more concordant segregation events. f CASR 755D found in salmon. g CASR 263S found in salmon. h CASR 465L found in Squalus sp. with a minor allele frequency of 0.04, they were not observed in 1136 normal Caucasian chromosomes (21). Similarly, the synonymous E870E variant is classified as uncommon; it was found in eight of 1136 Caucasian control chromosomes (21). FHH group The degree of hypercalcemia in the majority of FHH patients is similar to that of mild hyperparathyroidism. Most FHH patients demonstrate serum concentrations of PTH that are inappropriately normal, given the degree of hypercalcemia. About 20% of FHH patients have frankly elevated serum PTH levels. In contrast, the majority of PHPT patients have elevated serum PTH, but some have levels at or near the upper limit of normal, and there is overlap (28). With respect to comparative diagnostic indices, no single biochemical assay may be adequate for differentiating PHPT from FHH, although most cases will show discordance between calcium and PTH concentrations. Helpful features in the differential diagnosis are family history and identification of a renal calcium to creatinine clearance ratio that is less than 0.01, a feature rarely observed in PHPT (29). As emphasized here, identification of a CASR mutation provides key confirmatory evidence for the diagnosis. In the present study, sequence variants were detected in 53% (nine of 17) of the FHH patients. In seven of eight of the FHH kindreds, there was clear segregation of the mutation with affected status. Seven mutations were found in 11 FHH families having family members with hypercalcemia and/or hypocalciuria. This percentage (64%) is similar to those (60 66%) identified in previous studies (4 6). Lack of identification of a CASR mutation (e.g. family FHH-I) should not necessarily be taken as an exclusion because large parts of the gene (promoters and introns) were not examined in the present protocol. Also, FHH is genetically heterogeneous. Although most families maptothecasr gene at chromosome 3q , there is at least one family mapping to 19p13.3 (30) and another to 19q13 (31). PHPT group In the present series, CASR sequence variants were detected in four PHPT patients, but only one missense mutation was identified. In the vast majority of FHH cases, clinically overt hyperparathyroidism is not observed, although the present case can be classified with other examples in the recent literature (32 34). Whether this represents a coexistence of the two disorders or that the CASR mutation has contributed to the initiation or progression of PHPT is an ongoing debate. The overall low rate of identification of CASR mutations in the PHPT group highlights the power of the calcium to creatinine clearance ratio as a discriminatory factor in distinguishing between the two (35). FIHP group FIHP has been defined as hereditary primary hyperparathyroidism without the association of other disease or

9 J Clin Endocrinol Metab, April 2010, 95(4): jcem.endojournals.org 1827 tumors. However, some cases have been shown to be variants of other monogenic diseases: FHH, MEN1, or HPT-JT syndrome. In one FIHP kindred studied previously, affected individuals expressing hypercalcemia and hypercalciuria harbored a heterozygous CASR F881L mutation (18). Functional analysis in transfected kidney cells revealed a rather small (albeit significant) rightward shift in extracellular calcium/intracellular signaling response relative to the wild-type CASR. Because previous studies with the cultured parathyroid cells obtained from the patients of this family had indicated a more severe problem with respect to parathyroid secretory responsiveness (36), it was suggested that the mutation was likely to exert a tissue-selective effect and to be more deleterious in parathyroid than in kidney tubule. In one study of 36 FIHP kindreds, five affected individuals (14%) harbored heterozygous CASR mutations (19), and in another study of 22 unrelated FIHP subjects, four (15%) were identified as having heterozygous CASR mutations (20). In the present study, no CASR mutation was identified in any of the three FIHP families examined. Thus, although CASR mutations do contribute to FIHP overall, such cases are infrequent. Although MEN1 and HRPT2 mutations are also found in some FIHP kindreds, it is likely that other as yet unknown genes also contribute to this syndrome. Functional study Missense variants The in vitro functional analysis provided key information. The five CASR variants identified in the control subjects were no different from wild type. The finding of additional rare benign polymorphisms has implications for molecular diagnostic testing in that with a finding of a novel missense variant in a hypercalcemic individual in the absence of any other sequence change, it will remain uncertain as to whether the actual mutation has been identified. In vitro functional analysis established the deleterious nature of the missense and frameshift changes found in the FHH-affected individuals. The mutations are scattered throughout the CASR sequence and further emphasize that alterations in any one of a number of topologically distinct domains can lead to impairment of the receptor s activity. One can note that the I283T PHPT mutation, although clearly not having the activity of the wild type, was not as defective as the FHH mutations. This likely reflects the nature of the test system, the kidney cells, with the implication that the mutation might have more of an impact in the parathyroid chief cell than the kidney tubule. Splice-site mutation Whether the IVS4-19a3c variant affects splicing of the pre-mrna was assessed here by an in vitro minigene assay. Transfection with the minigene having the mutant CASR exon V insertion and flanking intron sequences led to two processed products, one representing normal, and the other aberrant, splicing. Although the minigene approach can provide important qualitative information with respect to the potential for a sequence change to affect splicing, quantitatively, the results do not always precisely mimic in vivo events (37 39). This may have to do with lack of required cis-acting elements in the gene sequences used and/or lack of needed trans-acting factors which may be deficient in the cell lines employed. However, in the present case, segregation of the sequence variant with FHH-affected family members, lack of the variant in any control chromosomes, and the in vitro functional analysis, all provide strong evidence that this is the disease-causing mutation. Bioinformatics Several predictive programs are being increasingly used to assign particular missense variants as being damaging or not. For the control subject group of the five missense variants, only one was predicted to be probably damaging in one of the three programs used, whereas the other four were benign. Although all four novel missense variants identified in the three FHH patients and one PHPT patient were classified as damaging in two of the three programs used, the third program predicted the mutants as being benign in three of the cases. The conclusion can be drawn that useful information is provided in most cases, but not all, and there is no substitute for functional analysis of each mutation. This caution is especially relevant to clinical genetics and the use of mutation identification for genetic counseling (40). Summary The study of this large cohort of controls and three patient groups with hypercalcemia has provided insights with respect to the clinical testing environment. Novel missense variants were found in control samples, and all had the same functionality as the wild-type receptor. For clinical molecular laboratories, reporting of such mutations should include some consideration that novel missense mutations have been observed in mutation surveys of healthy controls when there is uncertainty about the patient s clinical status (41). Conversely, it should be recognized that even careful clinical characterization of hypercalcemia and hypocalciuria in a single subject is not that likely to be associated with a presumptively causative CASR mutation unless there is a documented family his-

10 1828 Guarnieri et al. CASR Mutations in a Hypercalcemic Cohort J Clin Endocrinol Metab, April 2010, 95(4): tory. Even though the increased probability of finding such a mutation is statistically significant, at described here, there are FHH families for whom no mutation has been identified. CASR testing provides critical confirmatory evidence in the differential diagnosis of hypercalcemic states. Acknowledgments Address all correspondence and requests for reprints to: Geoffrey N. Hendy Ph.D., Calcium Research Laboratory, Room H4.67, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Quebec, Canada H3A 1A1. geoffrey.hendy@mcgill.ca. This work was supported by a grant from NSERC/Dairy Farmers of Canada (to D.E.C.C.), Canadian Institutes of Health Research Grants MOP and MOP (to G.N.H.), and grants from Ministero della Salute of Italy (Ricerca Corrente 2001, 2002, and 2008) (to A.S.), (Ricerca Corrente 2009) (to V.G.). 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