Calcium-sensing receptor mutations and denaturing high performance liquid chromatography

in Journal of Molecular Endocrinology
Authors:
David E C Cole
Search for other papers by David E C Cole in
Current site
Google Scholar
PubMed
Close
,
Francisco H J Yun
Search for other papers by Francisco H J Yun in
Current site
Google Scholar
PubMed
Close
,
Betty Y L Wong
Search for other papers by Betty Y L Wong in
Current site
Google Scholar
PubMed
Close
,
Andrew Y Shuen
Search for other papers by Andrew Y Shuen in
Current site
Google Scholar
PubMed
Close
,
Ronald A Booth
Search for other papers by Ronald A Booth in
Current site
Google Scholar
PubMed
Close
,
Alfredo Scillitani Departments of Laboratory Medicine and Pathobiology, Endocrinology Unit, Hormones and Cancer Research Unit, Medicine, and Genetics, University of Toronto, Ontario, Canada M5G 1L5

Search for other papers by Alfredo Scillitani in
Current site
Google Scholar
PubMed
Close
,
Svetlana Pidasheva Departments of Laboratory Medicine and Pathobiology, Endocrinology Unit, Hormones and Cancer Research Unit, Medicine, and Genetics, University of Toronto, Ontario, Canada M5G 1L5

Search for other papers by Svetlana Pidasheva in
Current site
Google Scholar
PubMed
Close
,
Xiang Zhou Departments of Laboratory Medicine and Pathobiology, Endocrinology Unit, Hormones and Cancer Research Unit, Medicine, and Genetics, University of Toronto, Ontario, Canada M5G 1L5

Search for other papers by Xiang Zhou in
Current site
Google Scholar
PubMed
Close
,
Lucie Canaff Departments of Laboratory Medicine and Pathobiology, Endocrinology Unit, Hormones and Cancer Research Unit, Medicine, and Genetics, University of Toronto, Ontario, Canada M5G 1L5

Search for other papers by Lucie Canaff in
Current site
Google Scholar
PubMed
Close
, and
Geoffrey N Hendy Departments of Laboratory Medicine and Pathobiology, Endocrinology Unit, Hormones and Cancer Research Unit, Medicine, and Genetics, University of Toronto, Ontario, Canada M5G 1L5

Search for other papers by Geoffrey N Hendy in
Current site
Google Scholar
PubMed
Close

Free access

Sign up for journal news

The calcium-sensing receptor (CASR), a plasma membrane G-protein-coupled receptor, is expressed in parathyroid gland and kidney, and controls systemic calcium homeostasis. Inactivating CASR mutations are associated with familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism, and activating mutations cause autosomal dominant hypocalcemia (ADH). CASR mutation identification plays an important role in the clinical management of mineral metabolism disorders. We describe here a high-throughput method using screening with denaturing high performance liquid chromatography (DHPLC) to initially interrogate 12 amplicons covering translated exons and exon/intron boundaries, followed by sequencing of any amplicon with a modified melting curve relative to wild type, and direct sequencing of a 13th amplicon encoding the COOH-terminal tail to distinguish causative mutations from three common missense single nucleotide polymorphisms. A blinded analysis of 32 positive controls representing mutations throughout the CASR sequence, as well as 22 negative controls, yielded a concordance rate of 100%. We report eight novel and five recurrent FHH mutations, along with six novel and two recurrent ADH mutations. Thus, DHPLC provides a rapid and effective means to screen for CASR mutations.

Abstract

The calcium-sensing receptor (CASR), a plasma membrane G-protein-coupled receptor, is expressed in parathyroid gland and kidney, and controls systemic calcium homeostasis. Inactivating CASR mutations are associated with familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism, and activating mutations cause autosomal dominant hypocalcemia (ADH). CASR mutation identification plays an important role in the clinical management of mineral metabolism disorders. We describe here a high-throughput method using screening with denaturing high performance liquid chromatography (DHPLC) to initially interrogate 12 amplicons covering translated exons and exon/intron boundaries, followed by sequencing of any amplicon with a modified melting curve relative to wild type, and direct sequencing of a 13th amplicon encoding the COOH-terminal tail to distinguish causative mutations from three common missense single nucleotide polymorphisms. A blinded analysis of 32 positive controls representing mutations throughout the CASR sequence, as well as 22 negative controls, yielded a concordance rate of 100%. We report eight novel and five recurrent FHH mutations, along with six novel and two recurrent ADH mutations. Thus, DHPLC provides a rapid and effective means to screen for CASR mutations.

Introduction

The calcium-sensing receptor (CASR) is a plasma membrane G-protein-coupled receptor (GPCR) abundantly expressed in the parathyroid gland and kidney tubule where it plays a key role as the ‘calciostat’, maintaining extracellular fluid calcium concentrations within a narrow normal range (Brown 2007). Activation of CASR by increased ionized calcium leads to decreased PTH secretion and increased calcium excretion. Naturally occurring CASR mutations contribute to perturbed blood calcium levels in several disease states. Hereditable hypercalcemic disorders such as familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT) can be caused by heterozygous and homozygous inactivating mutations in the CASR respectively (Pollak et al. 1993). Similarly, hypocalcemic disorders such as autosomal dominant hypocalcemia (ADH) can be associated with activating mutations in the receptor (Pollak et al. 1994).

The human CASR (MIM #601199) is encoded by six exons (exons 2–7) of the gene (Pollak et al. 1993, Pearce et al. 1995, Heath et al. 1996) located on chromosome 3q13.3-q21 (Janicic et al. 1995). Exons 1A and 1B encode alternative 5′ untranslated regions (5′-UTR) (Garrett et al. 1995, Chikatsu et al. 2000, Canaff & Hendy 2002), exon 2 encodes the common 5′-UTR, the ATG initiation codon, the signal peptide sequence, and the beginning of the extracellular domain (ECD); exons 3–6 encode the main part of the ECD; and exon 7 encodes the end of the ECD, the transmembrane domain (TMD), and the intracellular domain (ICD). The human receptor consists of 1078 amino acids, with ∼ 600 amino acids in the ECD, 250 in the TMD, and 216 in the cytoplasmic tail.

Over 100 mutations in the CASR have been described in association with FHH and/or NSHPT (Pidasheva et al. 2004). The majority are missense, with others being nonsense, insertion, and deletion/insertion. There are no mutation ‘hot spots’ but the mutations tend to cluster in two regions: the NH2-terminal 300 amino acids of the ECD; and a 340-residue stretch in the transmembrane and ICD (Hendy et al. 2000, see ). About 100 mutations in the CASR have been described in association with ADH (Pidasheva et al. 2004); most are missense while a handful in the intracellular tail are deletions. Activating mutations are more tightly clustered in a 150-amino acid stretch in the first NH2-terminal third of the ECD, and the latter half (∼100 amino acids) of the TMD. A few mutations are also found in the intracellular tail (Hendy et al. 2000, see ).

Various polymorphic variants have been identified in the CASR gene (Yun et al. 2007). Of note, three single nucleotide polymorphisms in exon 7 encode non-conservative amino acid changes (A986S, R990G, and Q1011E) in the COOH-terminal tail of the CASR protein. These polymorphisms have been found to be predictive of serum calcium concentrations in some normal Caucasian populations, either individually or in haplotype combination (Cole et al. 1999, 2001, Scillitani et al. 2004).

Identification of CASR mutations plays an important role in the clinical management of inherited hypercalcemic and hypocalcemic disorders. Positive CASR mutation testing is critical in differentiating FHH cases (in which the mild hypercalcemia is benign) from primary hyperparathyroidism (in which the hypercalcemia may be associated with disturbed bone and mineral metabolism). Thereby, the FHH individual may be spared an unnecessary parathyroidectomy. Assignment of a hypocalcemic individual as having an activating CASR mutation distinct from hypoparathyroidism of other etiologies alerts the physician to the special treatment requirements of such patients related to their over-activated renal CASR and greater risk of nephrocalcinosis or renal stones with vitamin D metabolite supplementation (see Hendy & Cole 2007).

Conventional CASR mutation searches begin with isolation of leukocyte DNA, followed by amplification of all six protein-coding exons and flanking intronic sequences and bidirectional sequencing of all amplicons. Alternative methods, e.g. RNase protection assay (Pollak et al. 1993, 1994) or pre-screening of amplicons by single-strand conformational polymorphism analysis (Pearce et al. 1995), have been reported in some studies but they have not achieved wide application. A newer technique that detects heteroduplexes in PCR amplicons by ion-pair reverse-phase high performance liquid chromatography has been developed (Oefner & Underhill 1998, Xiao & Oefner 2001). Denaturing high-performance liquid chromatography (DHPLC) has proved useful for mutational analysis of genes such as MEN1 (Crépin et al. 2006), RB1 (Houdayer et al. 2004), ENG and ALK-1 (Lenato et al. 2006), NPHS2/podocin (He et al. 2007), and BRCA1/2 (Gerhardus et al. 2007). We (Hendy et al. 2003) and others (Waller et al. 2004) have successfully utilized the DHPLC method for CASR mutational analysis in a few cases. Here, we present a detailed outline of the development and evaluation of a rapid throughput DHPLC protocol that allows amplicons of wild-type sequence to be screened out and only those showing evidence of heterozygosity selected for sequencing and mutation identification.

Materials and methods

Positive and negative control patient panels

Genomic DNA samples from 32 patients having either FHH/NSHPT or ADH and in whom CASR mutations/sequence variants (positive controls) had been previously identified by conventional (non-DHPLC) methods were subjected in a blinded fashion to DHPLC analysis. A further set of genomic DNA samples from 22 hypercalcemic or hypocalcemic individuals (presumptively FHH or ADH patients) in which no CASR mutations had been identified by conventional (non-DHPLC) analysis were examined by the DHPLC method. In addition, DHPLC analysis of selected samples from a study (Guarnieri et al. 2008) of a cohort of hypercalcemic patients (and controls) seen at the Endocrine Clinic, San Giovanni Rotondo Research Centre (Istituto di Ricovero e Cura a Carattere Scientifico, Casa Sollievo della Sofferenza, Italy) is presented here as representative of the utility of the method. FHH was diagnosed by asymptomatic hypercalcemia with high-normal or slightly elevated serum PTH levels. Diagnosis of NSHPT was based on marked hypercalcemia and serum PTH levels presenting shortly after birth. ADH was diagnosed by the finding of hypocalcemia with low or low-normal serum PTH levels. All subjects gave informed consent and studies were approved by institutional ethical committees.

Nucleic acid extraction and amplification

Genomic DNA was extracted from peripheral leukocytes using standard methods. The CASR protein-coding regions and intron/exon boundaries were PCR amplified as 13 fragments ranging in size from 202 to 455 bp (see Table 1 for specific primer sequences). For some primers, 5′ and 3′ GC clamp sequences were used to confer optimal melting profiles on each amplicon. Three basic GC clamp sequences supplied by Transgenomic (San Jose, CA, USA) (an 8-bp sequence (5′-gcgtcccg-3′), a 10-bp sequence (5′-gcccccgccg-3′), and a 20-bp sequence (5′-gcggcccgccgcccccgccg-3′)) were used either as is or shortened to produce an optimal melt profile as predicted by Wavemaker v. 4.1 software. Genomic DNA (2.5 ng/ml) was amplified in 1× PCR buffer (Qiagen), 0.2 mM of each dNTP, 0.5 mM of each primer, and 0.025 U/ml HotStarTaq (Qiagen) in a total volume of 20 μl. The thermal cycles comprised an initial DNA denaturation and HotStarTaq activation at 95 °C for 15 min, then 35 cycles of 94 °C for 20 s, annealing at 58–62 °C (see Table 1 for specific temperatures for each amplicon) for 20 s, and elongation at 72 °C for 20–25 s with an increment of 1 s after each cycle, and a final extension at 72 °C for 5 min. Samples were then annealed by initially heating to 95 °C for 5 min and cooling slowly to 30 °C over 30 min to allow heteroduplex formation. The samples were then either directly analyzed or stored at 4 °C before analysis by DHPLC.

Table 1

PCR primers and annealing temperature for each amplicon and denaturing high performance liquid chromatography conditions used

Size (bp)Primer sequencesaPCR annealing temperatureDHPLC initial %BElution temperature (°C)
Amplicon
E2305F: 5′-gcccccgccgCTCCTAGCTGTCTCATCCCTTG-3′ (5′UTR)b R: 5′-GTTTGGTGCAGCTTTCTCC-3′ (intronic)605360, 63
E3-1202F: 5′-gcgtcccgGCTTCCCATTTTCTTCCACTT-3′ (intronic) R: 5′-ACCAAACTCAGGGTGGCTTC-3′605059.5, 60.5
E3-2264F: 5′-gcccccgccgTTGCAACACCGTTTCTGAGG-3′ R: 5′-gcgtcccgGCCTGCTTCTTCTGATCCTG-3′ (intronic)605360.5, 62, 63
E4-1398F: 5′-gcccccgccgTCATTCACCATGTTCTTGGTTC-3′ (intronic) R: 5′-gcgtcccgCTTGATGAGGGGCTCAAGAT-3′605760.5, 61.5
E4-2377F: 5′-gcgtcccgCATGTGGTAGAGGTGATTCAAAA-3′ R: 5′-gcgtcccgGAAAGGTGTCCACAGGTAAAGG-3′605660.5, 61.5, 63
E4-3367F: 5′-TTTAACTGCCACCTCCAAGAAG-3′ R: 5′-GCAGCCCAACTCTGCTTTATT-3′ (intronic)605660, 61
E5343F: 5′-CAGGGCACAGCCTACCTAAT-3′ (intronic) R: 5′- CCTGGTGGAGACATCTGGTT-3′ (intronic) 605560
E6221F: 5′-gcgtcccgCTGGCCCCTGACCCTACAAC-3′ (intronic) R: 5′-gcgtcccgACAGTGCCCAAGAGGGGTTC-3′ (intronic)605162, 63
E7-1341F: 5′-gcccccgccgCACTCACACATTTTAGTCTGTGC-3′ (intronic) R: 5′- AAGAACAGGGAGCTGGAGAA-3′605562
E7-2284F: 5′-gcccgccgcccccgccgGCTCTCCTACCTCCTCCTCTTC-3′ R: 5′-gccgcccccgcccgGAAGGTGCAGAGGAAAACCA-3′605463.5, 64.5
E7-3414F: 5′-CTGGTGTTTGAGGCCAAGAT-3′ R: 5′-GATGGCAATCACCTCTACGG-3′585762, 63
E7-4432F: 5′-gcgtcccgCAGCCTATGCCAGCACCTAT-3′ R: 5′-cggcccgccgcccccgcccgCTGAGATCGTTGCTGCTGTG-3′625762, 65
E7-5455F: 5′-CTAACCCAGCAAGAGCAGCA-3′ R: 5′-TCTCCCTAGCCCAGTCTTCTC-3′ (3′UTR)60NAcNA

GC clamp sequences are lowercase and bolded.

Sequences are exonic except where indicated otherwise.

Amplicon E7-5 examined by sequence analysis not by DHPLC (see text).

DHPLC analysis

Heteroduplexes were resolved on a Transgenomic WAVE system equipped with a DNASep Cartridge column. Five microliters of PCR product were injected and eluted at the partial melting temperature with an incremental acetonitrile gradient of 9% in 4.5 min at a constant flow rate of 0.9 ml/min. The gradient was created by mixing buffer A (0.1 M triethylamine acetate buffer (TEAA), pH 7, 0.1 M Na2EDTA) with buffer B (25% acetonitrile, 0.1 M TEAA, and 0.1 M Na2EDTA); the initial concentrations of buffer B were adjusted according to the length of the fragment. After each analysis, the column was stripped with 100% acetonitrile for 0.5 min and regenerated in buffer B at 5% below the initial concentration for 1.5 min. The elution profile was monitored by u.v. absorbance at 260 nm.

DNA sequencing

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. Twenty microliters of each PCR product were purified using the QIAQuick PCR Purification kit (Qiagen) and eluted in 30 μl of buffer. The PCR products were directly sequenced in sense and antisense directions using the appropriate DHPLC–PCR primer set (but lacking any GC clamp sequences), using the ABI PRISM 3100-Avant Genetic Analyzer (Applied Biosystems, Foster City; CA, USA) and BigDye Terminator Cycle Sequencing Kit (v.1.1) according to the manufacturer's protocol. The E7-5 fragment, harboring the three common polymorphisms, A986S, R990G, and Q1011E was analyzed by DNA sequencing.

Bioinformatics

PolyPhen (PHEN –) () predicts the effect of an amino acid substitution on function based on knowledge of the protein's structure, interactions, and evolution (Sunyaev et al. 2001, Ramensky et al. 2002). The effect of missense CASR variants, classified as either probably damaging, possibly damaging or benign, was predicted from a position-specific independent counts score based on alignment of the sequences of known CASR orthologs in several species as well as of homologous group 3 GPCRs.

SIFT (Sorting Intolerant From Tolerant) () predicts whether a particular amino acid substitution would be tolerated or not tolerated (affect protein function) based on a homology analysis of the protein of interest (CASR) from several different species, as well as related proteins (group 3 GPCRs) (Ng & Henikoff 2003).

Results

DHPLC protocol optimization and validation

The entire CASR protein-coding sequence and the intron–exon boundaries were amplified as 13 fragments and the first 12 amplicons were subjected to DHPLC, while the 13th (E7-5 encoding three common polymorphisms) was subjected to direct sequencing (Fig. 1). The method was validated by the use of genomic DNA samples representing 32 CASR mutations/sequence variants (positive controls) previously identified by conventional (non-DHPLC) methods (Table 2). The positive controls were spread throughout the CASR-coding sequence, and covered most of the melting domains present in each fragment. Figure 1 details the relative position of each of the positive control mutations of the present study along with other reported mutations. Areas of high, medium, and low confidence of sequence variation detection are indicated. High confidence areas are those with optimal melt profiles and where analysis of positive controls, and/or detection of a sequence variant in an experimental sample (Guarnieri et al. 2008), provided confirmation of the capability to detect sequence alterations. Medium confidence areas are those with optimal melt profiles; however, no positive control or experimental samples were available within these areas. Low confidence areas do not have optimal melt profiles and no positive control or experimental samples were available. All sequence variations of the positive control samples were identified under conditions of at least one temperature, with the majority being detected at several analytical temperatures (Table 1).

Figure 1
Figure 1

Panel (A) genomic organization of the CASR that is comprised of eight exons. Untranslated regions, exons 1A and 1B, part of exon 2, and part of exon 7 are in gray, while part of exon 2, exons 3–6, and part of exon 7 that are protein-coding are in black. P1, promoter 1, P2, promoter 2. Panel (B) the 13 amplified CASR fragments that range in size from 202 to 455 bps (fragment sizes with GC clamps are shown in parentheses). Mutations reported in the CASR mutation database () and in the literature are indicated above the line: missense – inactivating (), activating (), unknown () – silent mutations (), insertion/deletion (,), and SNPs (). Mutations found through our in-house mutation screening are indicated as missense () or deletion (). Positive controls are presented as (). Note the large Alu insertion in E7-4 at codon 876 (red arrow) and the large deletion of codons 895–1075 overlapping E7-4 and E7-5 (blue arrow). Regions of high, satisfactory, and uncertain sensitivity are shown on blue, yellow, and pink backgrounds respectively (see text for details).

Citation: Journal of Molecular Endocrinology 42, 4; 10.1677/JME-08-0164

Table 2

Positive controls for denaturing high performance liquid chromatography (DHPLC) evaluation

MutationatypeAmpliconTrivial nameNucleotidebpositionNucleotide changeReferencesc
Numbers
1FHHE2-2C7fs-2X4719TGC >TTGCD'Souza-Li et al. (2002)
2FHHE2-2F42S125TTT > TCTNovel
3FHHE2-2P55L164CCG > CTGHeath et al. (1996)*
4FHHE3-1R66H197CGT > CATPidasheva et al. (2006)
5FHHE3-1I81M243ATA > ATGNovel
6ADHE3-2N118K354AAC > AAAPearce et al. (1996)*
7ADHE3-2L125F373CTT > TTTNovel
8ADHE3-2C129R385TGC>CGCNovel
9FHHE3-2T138M413ACG > ATGD'Souza-Li et al. (2002)
10FHHE3-2G143R427GGA>AGANovel
11FHHE3-2G158R472GGG > AGGNovel
12PolyE4-1IVS3+19492+19A>GYun et al. (2007)
13FHHE4-1S166G496AGT>GGTNovel
14FHHE4-1R220W658CGG > TGGD'Souza-Li et al. (2002)*
15ADHE4-1E228K682GAG > AAGNovel
16PolyE5S497S1491TCC > TCTYun et al. (2007)
17FHHE6G549R1645GGG > AGGD'Souza-Li et al. (2002)
18FHHE6C562Y1685TGC > TACBurski et al. (2002)*
19FHHE6C565G1693TGT >GGTNovel
20FHHE7-1C582Y1745TGT > TATPearce et al. (1995)*
21FHHE7-1N583X1747AAC > TAACPidasheva et al. (2006)
22ADHE7-1E604K1810GAG > AAGAlvarez-Hernandez et al. (2003)*
23FHHE7-1; E7-2C661Y1982TGC > TACNovel
24FHHE7-2R680H2039CGC > CACArunchaiva et al. (1998)*
25PolyE7-3P748P2244CCC > CCGYun et al. (2007)
26FHHE7-3I761del2281del ATGNovel
27FHHE7-3R795W2383CGG > TGGD'Souza-Li et al. (2002)
28ADHE7-3N802I2405AAT > ATTNovel
29ADHE7-3; E7-4G830S2488GGC > AGCNovel
30ADHE7-3; E7-4F832L2494TTT > CTT Novel
31ADHE7-3; E7-4F832S2495TTT > TCTDreimane et al. (2001)
32FHHE7-4C851fs-2X9812551ins CCAGD'Souza-Li et al. (2002)

FHH, familial hypocalciuric hypercalcemia; ADH, autosomal dominant hypocalcemia; Poly, polymorphism.

GenBank: X81086, the A of the initiation codon ATG is nucleotide +1.

Mutations are either previously identified by us or others (reference given) or novel. In some cases, the same mutation previously reported by another group has been identified by us in a different family or individual (reference with asterisk).

Optimization of the chromatographic conditions provided clear cut differences in the elution profiles of variant-containing and wild-type amplicons. For example, exon 4-2 amplicons harboring missense changes, T263M and I283T, or P274S, R285W or F351V, were clearly distinguished from wild type at 63 °C or 61.5 °C respectively (Figs 2 and 3). These particular mutations were identified in a study of a hypercalcemic cohort at a single endocrine clinic (Guarnieri et al. 2008), and DHPLC chromatographic profiles are included here to demonstrate the utility of the method.

Figure 2
Figure 2

DHPLC chromatograms for wild-type versus CASR variants (T263M and I283T) found in fragment E4-2, analyzed at 63.0 °C.

Citation: Journal of Molecular Endocrinology 42, 4; 10.1677/JME-08-0164

Figure 3
Figure 3

DHPLC chromatograms for wild-type versus CASR variants (P274S, R285W, and F351V) found in fragment E4-2, analyzed at 61.5 °C.

Citation: Journal of Molecular Endocrinology 42, 4; 10.1677/JME-08-0164

A panel of 22 negative controls comprising DNA samples from 15 hypercalcemic (presumptively FHH) and 7 hypocalcemic (presumptively ADH) patients, in which all protein-coding exons and intron/exon boundaries were of normal sequence by conventional (non-DHPLC) analysis, were confirmed as being of wild-type sequence by the DHPLC method.

Clinical and biochemical data

Of the FHH and ADH mutations represented in the positive control panel some have been previously published by us, some are recurrent (having been identified in other families or individuals), and some are novel (Table 2). The clinical and biochemical data available for these cases can be found as the supplementary data in the online version of the Journal of Molecular Endocrinology at .

Bioinformatics

We applied a bioinformatics analysis to the 16 FHH (inactivating-type) missense mutations in the panel (Table 3). All the amino acids involved (with one exception) are absolutely conserved among species. For all mutations, Polyphen predicted 11 to be damaging and 5 to be benign. SIFT predicted 12 to affect function (not tolerated) and 4 to be tolerated. There was discordance between the two programs for only 2 out of the 16 mutations. For six of the mutants, a previous in vitro analysis had been conducted demonstrating impairment relative to the wild-type CASR. Three (out of the six) were predicted by Polyphen to be damaging, whereas five were predicted by SIFT to affect function (Table 3).

Table 3

Bioinformatics analysis of FHH missense mutations

Trivial nameConservedbPHEN scorecSIFT scoredFunctionaleanalysisReferences
Numbersa
2F42S+00NNovel
3P55L++++YBai et al. (1996)
4R66H+00YPidasheva et al. (2006)
5I81M++++NNovel
9T138M+0+YBai et al. (1996)
10G143R++++NNovel
11G158R+*+0NNovel
13S166G+00NNovel
14R220W++++YD'Souza-Li et al. (2002)
17G549R+0+YD'Souza-Li et al. (2002)
18C562Y++++NBurski et al. (2002)
19C565G++++NNovel
20C582Y++++NPearce et al. (1995)
23C661Y+++NNovel
24R680H++++NArunchaiva et al. (1998)
27R795W++++YBai et al. (1996)

Mutation numbers in Table 2.

+, amino acid conserved in CASR of all species; +*, conserved except in Takifugu rubripes.

Score interpretation: 0, benign; +, possibly damaging; ++, probably damaging.

Score interpretation: 0, tolerated; +, affect function.

Functional analysis: N, No; Y, Yes (impaired function).

Discussion

Development and evaluation of the DHPLC protocols

While the CASR protein-coding mutations tend to cluster in the first 300 amino acids of the ECD and the TMD (TMD; amino acids 500–900), a comprehensive mutation-screening method encompassing all protein-coding exons and exon/intron junctions is required. The semi-automated DHPLC method described here provides a rapid and sensitive way of screening all CASR exons to select only those amplicons with a chromatographic profile which departs from wild type for further analysis by sequencing. A blinded evaluation of positive and negative controls, previously established by conventional sequencing, yielded a concordance rate of 100%. The high sensitivity of the DHPLC protocol is underscored by our previous success in demonstrating that the clinically unaffected mother of an ADH family was a mosaic for an activating CASR mutation (Hendy et al. 2003). In that instance, identification of the mutation in 5–10% of the individual's leukocyte DNA was not achieved by conventional analysis.

Further improvements to the present DHPLC method can be anticipated. As each novel mutation is catalogued, it is assessed as a potential positive control, both for saturation of all melting domains within the DHPLC amplicons, but also for sequencing. Eventually, they may be of unique value in a DHPLC quality assurance consortium of the sort described by Schollen et al. (2005). Also, at present, CASR sequence that includes the two promoters is not being interrogated and mutations in that region affecting gene transcription would be missed. The analysis of our set of positive controls (Table 2) clearly indicate that small deletions/insertions within protein-coding exons are readily detected, but larger insertions/deletions affecting the amplification step may not be so readily ascertained. To detect loss or gain of DNA representing an exon (or more) of the CASR gene, methods such as quantitative multiplex PCR of short fluorescent fragments (QMPSF – Houdayer et al. 2004) or multiplex ligation-dependent probe amplification (MLPA – Sellner & Taylor 2004) should be developed.

Novel and recurrent CASR mutations

The mutations in our positive control panel are either previously published or represent recurrent or novel mutations. Therefore, we are able to report here eight novel and five recurrent FHH mutations, and six novel and two recurrent ADH mutations. With one exception, all are missense. The majority of the new FHH mutations are within the ECD of the CASR (F42S, I81M, G143R, G158R, S166G, and C565G), with C661Y within TM-2 and I761del within extracellular loop-2. The scattering of mutations is consistent with the notion of CASR having multiple functional components that collectively contribute to activity and that a critical mutation in any one of them can cause major impairment (D'Souza-Li et al. 2002). The new ADH mutations are equally divided within the ECD (L125F, C129R, and E228K) and the intracellular loop-3, TM-7, extracellular loop-3 part of the TMD where ADH mutations are known to cluster (see (CASRdb)).

Bioinformatics

Predictive programs are increasingly used as adjuncts in the assessment of whether particular missense variants identified in molecular diagnostic testing are likely to negatively affect protein function. We had the opportunity to classify the 16 FHH mutants in the positive control panel as damaging or benign (PolyPhen), or not tolerated or tolerated (SIFT). While most of the mutations were flagged by both programs as potentially deleterious, a few were not so identified. These included (more so for PolyPhen than SIFT) CASR mutants that have been characterized by in vitro functional analysis as being impaired relative to wild type. While the programs provide insightful information in most cases, such information as relates to clinical genetics and genetic counseling should be used with caution (Tchernitchko et al. 2004).

Summary

In conclusion, use of the DHPLC method for CASR mutation searches is as effective as standard sequencing protocols, judging by the lack of discordance for a blind survey of positive and negative controls. DHPLC provides a rapid means to screen for CASR mutations in hypercalcemic families or individuals, and CASR testing provides a critical contribution to the differential diagnosis of hypercalcemic states.

Declaration of interest

None of the authors have any conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was supported by a grant from NSERC/Dairy Farmers of Canada (to D E C Cole), Canadian Institutes of Health Research Grants MOP-86581 and MOP-57730 (to G N Hendy), and grants from Ministero della Salute of Italy (Ricerca Corrente 2001, 2002, and 2008) (to A Scillitani). L Canaff was supported by a Kidney Foundation of Canada Biomedical Fellowship.

Acknowledgements

We thank the patients for their participation and physicians for providing clinical details and patient samples, and Bing Yang, Xiaoling Wang, Mei Li, and Irina Mosesova for their technical assistance.

References

  • Alvarez-Hernandez D, Santamaria I, Rodriguez-Garcia M, Iglesias P, Delgado-Lillo R & Cannata-Andia JB 2003 A novel mutation in the calcium-sensing receptor responsible for autosomal dominant hypocalcemia in a family with two uncommon parathyroid hormone polymorphisms. Journal of Molecular Endocrinology 31 255262.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Arunchaiva S, Pollak MR, Seidman CA, Pinhas-Hamiel O, Welch TR, Tsang RC & Smith EP 1998 Marked hypercalcemia in a five-month-old male associated with heterozygous point mutation in the calcium-sensing receptor gene. Program and Abstracts of the 80th Annual Meeting of the Endocrinology Society P3-535 495

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Bai M, Quinn SJ, Trivedi S, Kifor O, Pearce SHS, Pollak MR, Krapcho KJ, Hebert SC & Brown EM 1996 Expression and characterization of inactivating and activating mutations of the human Ca2+o-sensing receptor. Journal of Biological Chemistry 271 1953719545.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Brown EM 2007 Clinical lessons from the calcium-sensing receptor. Nature Clinical Practice. Endocrinology & Metabolism 3 122133.

  • Burski K, Torjussen B, Paulsen AQ & Bollerslev J 2002 Parathyroid adenona in a subject with familial hypocalciuric hypercalcemia: coincidence or causality? Journal of Clinical Endocrinology and Metabolism 87 10151016.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Canaff L & Hendy GN 2002 Human calcium-sensing receptor gene: vitamin D response elements in promoters P1 and P2 confer transcriptional responsiveness to 1,25-dihydroxyvitamin D. Journal of Biological Chemistry 277 3033730350.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Chikatsu N, Fukumoto S, Takeuchi Y, Suzawa M, Obara T, Matsumoto T & Fujita T 2000 Cloning and characterization of two promoters for the human calcium-sensing receptor (CaSR) and changes of CaSR expression in parathyroid adenomas. Journal of Biological Chemistry 275 75537557.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Cole DEC, Peltekova VD, Rubin LA, Hawker GA, Vieth R, Liew CC, Hwang DM, Evrovski J & Hendy GN 1999 A986S, polymorphism of the calcium-sensing receptor and circulating calcium concentrations. Lancet 353 112115.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Cole DEC, Vieth R, Trang HM, Wong BY-L, Hendy GN & Rubin LA 2001 Association between total serum calcium and the A986S polymorphism of the calcium-sensing receptor gene. Molecular Genetics and Metabolism 72 168174.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Crépin M, Pigny P, Escande F, Bauters CC, Calender A, Lefevre S, Buisine M-P, Porchet N & Odou M-F 2006 Evaluation of denaturing high performance liquid chromatography for the mutational analysis of the MEN1 gene. Journal of Molecular Endocrinology 36 369376.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Dreimane D, Hendy GN, Alon U & Geffner M 2001 Normalization of serum calcium, phosphorus, and magnesium with homeopathic PTH in a child with hypocalcemic hypercalciuria (HCHC) and a mutation of the calcium-sensing receptor gene. Program and Abstracts of the 80th Annual Meeting of the Endocrinology Society P3-125 475

    • PubMed
    • Search Google Scholar
    • Export Citation
  • D'Souza-Li L, Yang B, Canaff L, Bai M, Hanley DA, Bastepe M, Salisbury SR, Brown EM, Cole DEC & Hendy GN 2002 Identification and functional characterization of novel calcium-sensing receptor mutations in familial hypocalciuric hypercalcemia and autosomal dominant hypocalcemia. Journal of Clinical Endocrinology and Metabolism 87 13091318.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Garrett JE, Capuano IV, Hammerland LG, Hung BC, Brown EM, Hebert SC, Nemeth EF & Fuller F 1995 Molecular cloning and functional expression of human parathyroid calcium receptor cDNAs. Journal of Biological Chemistry 270 1291912925.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Gerhardus A, Schleberger H, Schlegelberger B & Gadzicki D 2007 Diagnostic accuracy of methods for the detection of BRCA1 and BRCA2 mutations: a systematic review. European Journal of Human Genetics 15 619627.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Guarnieri V, Scillitani A, Battista C, Muscarella LA, Coco M, D'Agruma L, Sacco M, Canaff L, Yun F, Wong BYL, Hendy GN & Cole DEC 2008 Molecular diagnostic strategies for mutations of the calcium-sensing receptor gene: application to a large cohort of patients with different hypercalcemic syndromes. Journal of Bone and Mineral Research 23 423

    • PubMed
    • Search Google Scholar
    • Export Citation
  • He N, Zahirieh A, Mei Y, Lee B, Senthilnathan S, Wong B, Mucha B, Hildebrandt F, Cole DEC & Cattran D et al. 2007 Recessive NPHS2 (podocin) mutations are rare in adult-onset idiopathic focal segmental glomerulosclerosis. Clinical Journal of the American Society of Nephrology 2 3137.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Heath H III, Odelberg S, Jackson CE, Teh BT, Hayward N, Larsson C, Buist NR, Krapcho KJ, Hung BC & Capuano IV et al. 1996 Clustered inactivating mutations and benign polymorphisms of the calcium receptor gene in familial benign hypocalciuric hypercalcemia suggest receptor functional domains. Journal of Clinical Endocrinology and Metabolism 81 13121317.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Hendy GN & Cole DEC 2007 Parathyroid Disorders. In Emery and Rimoin's Principles and Practice of Medical Genetics 5th edition, vol 2 ch 89, pp 1951–1979. Eds DL Rimoin, JM Connor, RE Pyeritz & BE Korf. Edinburgh: Churchill Livingstone.

    • PubMed
    • Export Citation
  • Hendy GN, D'Souza-Li L, Yang B, Canaff L & Cole DEC 2000 Mutation update, mutations in the calcium-sensing receptor (CASR) in familial hypocalciuric hypercalcemia, neonatal severe hyperparathyroidism and autosomal dominant hypocalcemia. Human Mutation 16 281296.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Hendy GN, Minutti C, Canaff L, Pidasheva S, Yang B, Nouhi Z, Zimmerman D, Wei C & Cole DEC 2003 Recurrent familial hypocalcemia due to germline mosaicism for an activating mutation of the calcium-sensing receptor gene. Journal of Clinical Endocrinology and Metabolism 88 36743681.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Houdayer C, Gauthier-Villars M, Lauge A, Pagès-Berhouet S, Dehainault C, Caux-Moncoutier V, Karczynski P, Tosi M, Doz E & Desjardins L et al. 2004 Comprehensive screening for constitutional RB1 mutations by DHPLC and QMPSF. Human Mutation 23 193202.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Janicic N, Soliman E, Pausova Z, Seldin MF, Rivière M, Szpirer J, Szpirer C & Hendy GN 1995 Mapping of the Ca2+-sensing receptor gene to human chromosome 3q13.3-21 by fluorescence in situ hybridization, and localization to rat chromosome 11 and mouse chromosome 16. Mammalian Genome 6 798801.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Law WM & Heath H III 1985 Familial benign hypercalcemia (hypocalciuric hypercalcemia): clinical and pathogenetic study of 21 families. Annals of Internal Medicine 102 511519.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Lenato GM, Lastella P, Di Giacomo MC, Resta N, Suppressa P, Pasculli G, Sabbà C & Guanti G 2006 DHPLC-based mutations analysis of ENG and ALK-1 genes in HHT Italian populations. Human Mutation27 213–214.

    • PubMed
    • Export Citation
  • Marx SJ, Attie MF, Levine MA, Spiegel AM, Downs RWJ & Lasker RD 1981 The hypocalciuric or benign variant of familial hypercalcemia: clinical and biochemical features of fifteen families. Medicine 60 397412.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ng PC & Henikoff S 2003 SIFT: predicting amino acid changes that affect protein function. Nuclear Receptor Signaling 31 38123814.

  • Oefner PJ & Underhill PA 1998 DNA Mutation Detection Using Denaturing High-performance Liquid Chromatography (DHPLC) . In Current Protocols in Human Genetics , vol 19:7.10.1-7.10.12 , Eds Dracopoli NC, Haines JL, Korf BR, Moir DT, Morton CC, Seidman CE, Seidman JG, Smith DR . New York: Wiley & Sons.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Pearce SHS, Trump D, Wooding C, Besser GM, Chew S, Heath D, Hughes I & Thakker RV 1995 Calcium-sensing receptor mutations in familial benign hypercalcemia and neonatal hyperparathyroidism. The Journal of Clinical Investigation 96 26832692.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Pearce SHS, Williamson C, Kifor O, Bai M, Coulthard MG, Davies M, Lewis-Barned N, McCredie D, Powell H & Kendall-Taylor P et al. 1996 A familial syndrome of hypocalcemia with hypercalciuria due to mutations in the calcium-sensing receptor. New England Journal of Medicine 335 11151122.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Pidasheva S, D'Souza-Li L, Canaff L, Cole DEC & Hendy GN 2004 CASRdb, calcium-sensing receptor locus-specific database for mutations causing familial (benign) hypocalciuric hypercalcemia, neonatal severe hyperparathyroidism and autosomal dominant hypocalcemia. Human Mutation 24 107111.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Pidasheva S, Grant M, Canaff L, Ercan O, Kumar U & Hendy GN 2006 The calcium-sensing receptor (CASR) dimerizes in the endoplasmic reticulum: biochemical and biophysical characterization of novel CASR mutations causing familial hypocalciuric hypercalcemia. Human Molecular Genetics 15 22002209.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Pollak MR, Brown EM, Chou Y-HW, Hebert SC, Marx SJ, Steinmann B, Levi T, Seidman CE & Seidman JG 1993 Mutations in the human Ca2+-sensing receptor gene cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Cell 75 12971303.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Pollak MR, Brown EM, Estep HL, McLaine PN, Kifor O, Park J, Hebert SC, Seidman CE & Seidman JG 1994 Autosomal dominant hypocalcemia caused by a Ca2+-sensing receptor gene mutation. Nature Genetics 8 303307.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ramensky V, Bork P & Sunyaev S 2002 Human non-synonymous SNPs: server and survey. Nuclear Receptor Signaling 30 38943900.

  • Schollen E, Dequeker E, McQuaid S, Vankeirsbilck B, Michils G, Harvey J, van den Akker E, van Schooten R, Clark Z & Schrooten S et al. 2005 DDQA, Collaborative Group Diagnostic DHPLC Quality Assurance (DDQA): a collaborative approach to the generation of validated and standardized methods for DHPLC-based mutation screening in clinical genetics laboratories. Human Mutation 25 583592.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Scillitani A, Guarnieri V, De Geronimo S, Muscarella LA, Battista C, D'Agruma L, Bertoldo F, Florio C, Minisola S & Hendy GN et al. 2004 Blood ionized calcium is associated with clustered polymorphisms in the carboxy-terminal tail of the calcium-sensing receptor. Journal of Clinical Endocrinology and Metabolism 89 56345638.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Sellner LN & Taylor GR 2004 MLPA and MAPH: new techniques for detection of gene deletions. Human Mutation 23 413419.

  • Sunyaev S, Ramensky V, Koch I, Lathe W III, Kondrashov AS & Bork P 2001 Prediction of deleterious human alleles. Human Molecular Genetics 10 591597.

  • Tchernitchko D, Goossens M & Wajcmen H 2004 Insilico prediction of the deleterious effect of a mutation: proceed with caution in clinical genetics. Clinical Chemistry 50 19741978.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Waller S, Kurzawinski T, Spitz L, Thakker R, Cranston T, Pearce S, Cheetham T & van't Hoff WG 2004 Neonatal severe hyperparathyroidism: genotype/phenotype correlation and the use of pamidronate as rescue therapy. European Journal of Pediatrics 163 589594.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Xiao W & Oefner PJ 2001 Denaturing high-performance liquid chromatography: A review. Human Mutation 17 439474.

  • Yun FHJ, Wong BYL, Chase M, Shuen AY, Canaff L, Thongthai K, Siminovitch K, Hendy GN & Cole DEC 2007 Genetic variation at the calcium-sensing receptor (CASR) locus: Implications for clinical molecular diagnostics. Clinical Biochemistry 40 551561.

    • PubMed
    • Search Google Scholar
    • Export Citation

Supplementary Materials

 

  • Collapse
  • Expand
  • Panel (A) genomic organization of the CASR that is comprised of eight exons. Untranslated regions, exons 1A and 1B, part of exon 2, and part of exon 7 are in gray, while part of exon 2, exons 3–6, and part of exon 7 that are protein-coding are in black. P1, promoter 1, P2, promoter 2. Panel (B) the 13 amplified CASR fragments that range in size from 202 to 455 bps (fragment sizes with GC clamps are shown in parentheses). Mutations reported in the CASR mutation database () and in the literature are indicated above the line: missense – inactivating (), activating (), unknown () – silent mutations (), insertion/deletion (,), and SNPs (). Mutations found through our in-house mutation screening are indicated as missense () or deletion (). Positive controls are presented as (). Note the large Alu insertion in E7-4 at codon 876 (red arrow) and the large deletion of codons 895–1075 overlapping E7-4 and E7-5 (blue arrow). Regions of high, satisfactory, and uncertain sensitivity are shown on blue, yellow, and pink backgrounds respectively (see text for details).

  • DHPLC chromatograms for wild-type versus CASR variants (T263M and I283T) found in fragment E4-2, analyzed at 63.0 °C.

  • DHPLC chromatograms for wild-type versus CASR variants (P274S, R285W, and F351V) found in fragment E4-2, analyzed at 61.5 °C.