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amplification-created restriction site techniques. Total calcium, ionized calcium and urine calcium excretion were measured using automated clinical chemistry.

Original Article Serum calcium, urine calcium and polymorphisms of the calcium sensing receptor gene Claire Kelly1, Ian R Gunn1, Dairena Gaffney2 and Manjit S Devgun1

Abstract Addresses 1 Biochemistry Department, Wishaw General Hospital, Lanarkshire ML2 0DP, UK; 2 DNA Laboratory, Biochemistry Department, Royal Infirmary, Glasgow, Scotland, UK Correspondence Claire Kelly E-mail: [email protected]

Background Recent studies have suggested a correlation between the A986S polymorphism of the calcium sensing receptor (CASR), and serum total and ionized calcium. This study aimed to assess the prevalence of three CASR polymorphisms in a West of Scotland population and relate genotype to serum and urine calcium levels. Methods Fasting blood and urine samples were obtained from 121 healthy male and female volunteers aged 20–60 years. Volunteers were genotyped for the A986S, Q1011E and R990G polymorphisms using allele-specific amplification and amplification-created restriction site techniques. Total calcium, ionized calcium and urine calcium excretion were measured using automated clinical chemistry analysers. Results Genotype frequencies for the A986S polymorphism were: AA, 74.4%; AS, 24.8%; SS, 0.8%. There was a small but statistically significant (Po0.01) increase in ionized calcium concentration in AS individuals compared with the wild type (1.22 versus 1.20 mmol/L). No statistical difference was detected in serum total calcium or parameters of urine calcium excretion. Genotype frequencies for the remaining polymorphisms were: RR, 82.6%; RG, 16.5%; GG, 0.8% and QQ, 93.4%; QE, 6.6%; EE, 0%. Biochemical parameters in these individuals were not statistically different from the wild type. Conclusion The increase in serum ionized calcium in the AS group was small and, therefore, unlikely to be of clinical significance. Ann Clin Biochem 2006; 43: 503–506

Introduction The calcium-sensing receptor (CASR) is a 120-kDa glycoprotein made up of 1078 amino acids and belongs to family C of the G-protein-coupled receptor (GPCR) superfamily. In 1996, Heath et al.1 were the ¢rst to report the existence of three apparently benign polymorphisms located in the COOH-terminal of the CASR protein. These polymorphisms were serendipitously discovered while investigating 14 families with familial benign hypocalciuric hypercalcaemia (FBHH) for the presence of inactivating mutations, which result in an increase in the set point of the parathyroid cells resulting in a higher than normal extracellular ionized calcium requirement to suppress parathyroid hormone (PTH) release and increase calcium excretion by the kidneys. r 2006 The Association for Clinical Biochemistry

The polymorphisms, 986 Ala/Ser (A986S), 990 Gly/ Arg (G990R) and 1011 Gln/Glu (Q1011E), were located in exon seven of the CASR gene and they encode nonconservative exchanges of single amino acids. The S allele of the variant 986S polymorphism was found to be the most frequent of the three, with 30% of the population found to be heterozygous. Rare allele frequencies of the other two were G990R, 15% and Q1011E,10%. In1999, Cole et al.2 described a signi¢cant correlation between the A986S variant and increased serum calcium. This raised the possibility that subjects with the variant might be labelled hypercalcaemic and misdiagnosed as primary hyperparathyroid. Our study aimed at investigating the prevalence of the CASR polymorphisms in the West of Scotland population, relating genotypes to total calcium, 503

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ionized calcium and urine calcium excretion and assessing whether any di¡erences found might be likely to cause diagnostic uncertainty in cases of mild hypercalcaemia.

Methods Volunteers, comprising 121 healthy individuals (89 women and 32 men), between 20 and 60 years were recruited from sta¡ at our local hospital. Excluded from the study were those individuals reporting ethnicity other than Caucasian and those taking any of the following medications: calcium (41000 mg per day), vitamin D (420 mg per day or 4800 IU per day), thiazide diuretics, furosemide, calcium channel blockers, lithium and steroid hormones. For DNA preparation and biochemical studies, a 20 mL peripheral venous blood sample was taken, and a 20 mL second void urine sample was collected following a 12-h overnight fast. All blood samples were analysed for serum total calcium, ionized calcium, albumin, creatinine, parathyroid hormone and typed for the three polymorphisms described. Urine specimens were analysed for calcium and creatinine. All participants gave informed written consent, and the local Research Ethics Committee approved the study.

DNA extraction Leucocyte DNA was prepared from EDTA anticoagulated blood using the small-scale salting method essentially as described by Olerup and Zetterquist.3

DNA analysis Genotyping the A986S allele was performed using the allele-speci¢c ampli¢cation technique described by Cole et al.4 An analogous method using the following primers was devised to detect the Q1011E polymorphism: CSRQ1011Ecom:50 -AGC GAT ACG CTG ACC CGA CAC C-30 CSRQ1011Erare:50 -CCT GGA GGC CCA GAA AAG Cga CGA TAC GCT GAC CCG ACA CG-30 Non-complementary nucleotides introduced to minimize heteroduplexing are indicated in lower case, and allele-speci¢c complementary nucleotides are indicated in bold. The reverse primer was as described by Cole et al.4 for the detection of the A986S allele. Ampli¢cation-created restriction site (ACRS) analysis was used for the detection of the R990G polymorphism. A primer was used to deliberately substitute an alanine residue for a cytosine residue (shown in lower case). The result was the creation of an SmaI restriction Ann Clin Biochem 2006; 43: 503–506

site only when the 990G allele was present. Once again the same reverse primer was used. CSRR990Gfor:50 -CTC AGA AGA ACG CCA TGG CCC cC-30 The PCR procedure was as described by Cole et al.4 with an annealing temperature of 611C. DNA products were digested with SmaI (New England BioLabs) according to the manufacturer’s instructions and fragments separated on an 8% polyacrylamide gel in Tris--borate--EDTA bu¡er and visualized under UV transillumination following staining with ethidium bromide.

Biochemical analysis All biochemical analysis of serum and urine samples was carried out on automated clinical chemistry analysers. Serum total calcium (coe⁄cient of variation [CV] o2.17% at 2.30 mmol/L) was measured by an indirect ISE method (Beckman LX20), while free serum ionized calcium (CV o1.68% at 1.19 mmol/L) was measured using a calcium ion selective electrode (Bayer 855). Serum samples were obtained only when immediate analysis was possible; however, ionized calcium data corrected to pH7.4 were also collected. Serum albumin estimation (CVo2.81% at 36 g/L) was carried out by a bromocresol green method (Beckman LX20), and a rate Ja¡e method (Beckman LX20) was used to measure serum creatinine (CV o 4.95% at 101 mmol/L) and urine creatinine (CV o2.7% at 16.7 mmol/L). PTH analysis (CVo 8.93% at 2.24 pmol/L) was performed on an Elecsys 1010 Roche analyser, and urine calcium analysis (CV o3.25% at 3.7 mmol/L) was performed on an atomic absorption spectrophotometer (Perkin Elmer Analyst 100).

Statistical analysis For the purpose of analysing biochemical data, the AA/ QQ/RR genotype was considered to be ‘wild-type’ (i.e., homozygous common allele), and all other genotypes were compared with this group. This study was designed to have an 80% power of detecting a 0.06 mmol/L di¡erence in total serum calcium concentration between groups. Di¡erences in biochemical data relative to genotype were tested for statistical signi¢cance using unpaired two-sided t-tests assuming unequal variances, and a P-value less than 0.05 was deemed signi¢cant.

Results Table 1 shows the genotype distribution for all three CASR polymorphisms studied. The A986S was shown to be the most frequent polymorphism with 25% of the

Serum calcium, urine calcium and the polymorphisms of the CASR gene

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Table 1 Genotype distribution Homozygous wild

A986S R990G Q1011E

Heterozygous

Homozygous rare

Genotype

No. (%)

Genotype

No. (%)

Genotype

No. (%)

AA RR QQ

90 (74) 100 (83) 113 (93)

AS RG RE

30 (25) 20 (16) 8 (7)

SS GG EE

1 (1) 1 (1) 0

Table 2 Biochemical parameters

Ionized calcium (mmol/L) Total calcium (mmol/L) Adjusted calcium (mmol/L)w PTH (pmol/L) Urine calcium/creatinine ratio (mmol:mmol)

AA (n=90) Mean (SD)

AS (n=30) Mean (SD)

SS (n=1)

1.20 2.32 2.38 3.9 0.20

1.22 2.32 2.4 3.9 0.19

1.24 2.37 2.41 3.4 0.25

(0.05)* (0.10) (0.09) (1.02) (0.09)

(0.04)* (0.09) (0.09) (1.30) (0.1)

*Po0.01 between these groups. All other differences were not significant Adjusted calcium=measured calcium+(0.02  (47albumin)) mmol/L

w

population found to be heterozygous, followed by the RG genotype (16%) and the QE genotype (7%). Table 2 illustrates that AS individuals had an ionized calcium concentration statistically higher than that of wild-type individuals. Biochemical parameters for all other genotypes studied were not statistically di¡erent from those of wild-type subjects. There were no statistically signi¢cant di¡erences in any biochemical parameters between women and men and between age groups 21--40 and 41--60 although this study was not powered to detect any small di¡erences. The lowest ionized calcium concentration (1.16 mmol/L with a PTH concentration of 4.3 pmol/L) was observed in the only individual expressing the AA/QQ/GG genotype. The highest ionized calcium (1.43 with a PTH concentration of 10.0 pmol/L) was observed in the single subject found to express both the Q1011E and the R990G polymorphisms. The signi¢cance of these results cannot be con¢rmed due to the single observations and, therefore, further investigation would be required to assess whether these rare genotypes result in consistent alterations in ionized calcium.

Discussion In terms of genotype frequency, the occurrence of A986S wild types, heterozygotes- and homozygoterare (74. 4%, 24.8% and 0.8%, respectively) individuals in the West of Scotland appears to be comparable with studies in other Caucasian populations, e.g. Canadian (70.5%, 26.4% and 3.1%),2 New Zealand (71.6%, 23.5% and 4.9%)5 and Swedish (71.1%, 25.8% and 3.1%).6

These studies show the A986S polymorphism to be the most prevalent variant in Caucasian populations. In terms of the other two polymorphisms, in the Q1011E polymorphism we found frequencies (93.4% QQ, 6.6% QE, 0% EE) similar to those ¢rst described by Heath et al.,1 and, like many other studies, we report the R990 allele to be the more frequent of the two (82.6% RR, 16.5% RG, 0.8% GG). Kanazawa et al.7 have shown the A986S variant to be extremely rare in the Japanese population, while the 990G variant appears to be far more frequent in the Asian population.8 Similarly in Afro-Americans the A986S polymorphism appears to be very rare, while the Q1011E has a much higher prevalence.8 This study shows a small but statistically signi¢cant increase in ionized calcium in those individuals heterozygous for the A986S polymorphism. These ¢ndings are in agreement with those of Cole et al.2 who studied a large group of healthy Canadian female adults, and Lorentzon et al.6 whose subjects were healthy adolescent girls. Several studies have failed to ¢nd such a correlation. One such group speci¢cally investigated postmenopausal women and a possible link between CASR polymorphisms and osteoporosis. They concluded that in these women there was no relationship between the polymorphism and serum calcium, baseline bone mineral density (BMD) or BMD decline.5 Similarly, a study which aimed to investigate the prevalence of the A986S polymorphisms in patients with hyperparathyroidism found no signi¢cant correlation between serum calcium levels and the A986S variant, but did report that patients with the QE genotype showed signi¢cantly higher total serum calcium concentrations and PTH levels.9 Ann Clin Biochem 2006; 43: 503–506

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Discrepancies in the literature on this subject to date may be the result of variances in ethnic and genetic backgrounds. However, Type I and Type II statistical errors may also play a part. This may certainly be true for the homozygote rare variants of all three polymorphisms whose frequency is less than 5% for all studies to date. Our study was powered to detect signi¢cant di¡erences in serum calcium but not urine calcium. This study found the prevalence of the A986S, Q1011E and R990G polymorphisms in the West of Scotland population to be similar to those frequencies previously described for Caucasian populations. The increase in serum-ionized calcium in the AS group, although statistically signi¢cant, was marginal and therefore unlikely to be of clinical signi¢cance.

References 1 Heath H, Odelberg S, Jackson C, et al. Clustered inactivating mutations and benign polymorphisms of the calcium receptor gene in familial benign hypocalciuric hypercalcemia suggest receptor function domains. J Clin Endocrinol Metab 1996; 81: 1312–17 2 Cole D, Peltekova V, Rubin L, et al. A986S polymorphism of the calcium-sensing receptor and circulating calcium concentrations. Lancet 1999; 353: 112–15 3 Olerup O, Zetterquist H. HLA-DR typing by PCR amplification with sequence-specific primers (PCR-SSP) in 2 hours: an alternative to

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serological DR typing in clinical practice including donor–recipient matching in cadaveric transplantation. Tissue Antigens 1992; 39: 225–35 Cole D, Vieth R, Trang H, et al. Association between total serum calcium and the A986S polymorphism of the calcium-sensing receptor gene. Mol Genet Metab 2001; 72: 168–72 Young R, Wu F, Van De Water N, Ames R, Gamble G, Reid I. Calcium sensing gene A986S polymorphism and the responsiveness to calcium supplementation in postmenopausal women. J Clin Endocrinol Metab 2003; 88: 697–700 Lorentzon M, Lorentzon R, Lerner U, Nordstrom P. Calcium sensing receptor gene polymorphism, circulating calcium concentrations and bone mineral density in healthy adolescent girls. Eur J Endocrinol 2001; 144: 257–61 Kanazawa H, Tanaka H, Kodama S, Morlwake A, Kobayashi M, Seino Y. The effect of calcium-sensing receptor gene polymorphisms on serum calcium levels. A familial hypocalciuric hypercalcemia family without mutation in the calcium-sensing receptor gene. Endocr J 2000; 47: 29–35 Hendy GN, D’Souza-Li L, Yang B, Canaff L, Cole DEC. Mutations of the calcium-sensing receptor (CASR) in familial hypocalciuric hypercalcaemia, neonatal severe hyperparathyroidism and autosomal dominant hypocalcemia. Hum Mutat 2000; 16: 281–96 Miedlich S, Lamesch P, Paschkle R. Frequency of the calciumsensing receptor variant A986S in patients with primary hyperparathyroidism. Eur J Endocrinol 145: 421–27

Accepted for publication 19 July 2006

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