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Recurrent Familial Hypocalcemia Due to Germline Mosaicism for an Activating Mutation of the Calcium-Sensing Receptor Gene

Departments of Medicine, Human Genetics, and Physiology, McGill University and Calcium Research Laboratory, Royal Victoria Hospital (G.N.H., L.C., S.P., B.Y., Z.N.), Montréal, Québec, Canada H3A 1A1; Department of Pediatrics, Mayo Clinic (C.M., D.Z.), Rochester, Minnesota 55905; and Departments of Laboratory Medicine and Pathobiology, Medicine, and Genetics, University of Toronto and The Banting Institute (C.W., D.E.C.C.), Toronto, Ontario, Canada M5G 1L5

Address all correspondence and requests for reprints to: Dr. G. N. Hendy, Calcium Research Laboratory Royal Victoria Hospital, Room H4.67, 687 Pine Avenue West, Montréal, Québec, Canada H3A 1A1. E-mail: geoffrey.hendy{at}mcgill.ca.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
De novo activating mutations in the calcium-sensing receptor (CASR) gene are a common cause of sporadic isolated hypoparathyroidism. Here, we describe a family in which two affected siblings were found to be heterozygous for a novel F788L mutation in the fifth transmembrane domain encoded by exon 7 of the CASR. Both parents and the third sibling were clinically unaffected and genotypically normal by direct sequencing of their leukocyte exon 7 PCR amplicons. However, the mother was revealed to be a mosaic for the mutation by sequence analysis of multiple subclones as well as denaturing HPLC of the CASR exon 7 leukocyte PCR product. A functional analysis of the mutation was performed by transiently transfecting wild-type and mutant CASRs tagged with a c-Myc epitope in human embryonic kidney (HEK293) cells. The mutant CASR was expressed at a similar level as the wild type. The F788L mutant produced a significant shift to the left relative to the wild-type CASR in the MAPK response to increasing extracellular calcium concentrations. This is the first report of mosaicism for an activating CASR mutation and suggests that care should be exercised in counseling for risks of recurrence in a situation where a de novo mutation appears likely.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
THE CALCIUM-SENSING receptor (CASR) is a plasma membrane G protein-coupled receptor expressed in the parathyroid gland and the cells lining the kidney tubule (1, 2). By virtue of its ability to sense small changes in circulating calcium concentration and couple this information to intracellular signaling pathways that modify PTH secretion or renal cation handling, this receptor plays an essential role in maintaining mineral ion homeostasis. Inherited abnormalities of the CASR gene located on chromosome 3q13.3–21 (3) can cause either hypercalcemia or hypocalcemia depending on whether they are inactivating (4) or activating (5), respectively.

Gain of function mutations in the CASR gene have been identified in several families previously diagnosed with autosomal dominant hypocalcemia (ADH; MIM#601198) (6), autosomal dominant hypoparathyroidism, and hypocalcemic hypercalciuria (7, 8, 9, 10). In the parathyroid gland the activated CASR suppresses PTH secretion, and in the kidney it induces hypercalciuria, which contributes to the hypocalcemia. Because of the marked hypercalciuria, there is a risk of other renal complications, such as nephrocalcinosis, renal stones, and impaired renal function (11). Renal tubular cells, excessively inhibited from absorbing calcium by the overactive CASR, sustain the hypercalciuria. Thus, considerable caution should be exercised to avoid overtreatment of ADH with vitamin D or calcium supplements.

In most cases of ADH, a family history is clear, but examples of de novo mutation are well known (7). Estimates of up to 47% have been suggested for the presence of a CASR mutation in cases of sporadic hypoparathyroidism (12, 13). Here, we report a family in which two affected siblings were found to carry a single novel mutation in the CASR gene. Although clinically unaffected and genotypically normal by standard molecular genetic analysis, we show, by more sensitive methods, that the mother is mosaic for the mutation.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Family K

This family came to clinical attention because of symptomatic hypocalcemia in individual III-2 (Fig. 1). Although there is no history of hypocalcemia on either side of the family, there is an uncertain report of kidney stones in the paternal grandfather (I-1, deceased). The maternal grandfather (I-3) and grandmother (I-4) have no history of kidney stones, nor do any of their siblings. The father of the index case (II-1) has a history of nephrolithiasis, but has normal levels of serum total calcium and phosphate and a normal 24-h urinary calcium/creatinine clearance ratio (see Table 1). The mother (II-2) also has a history of kidney stones with serum total calcium toward the lower limit of normal, but a normal 24-h urinary calcium/creatinine clearance ratio. She describes intermittent hoarseness, but no tingling or cramps. An older son (III-1) is normal. The index case (III-2) presented at 6 6/12 yr with headaches, decreased energy, muscle cramps, postexertional laryngeal stridor, diplopia, and visual blurring. A computed tomographic scan of the head showed calcification of the basal ganglia and the cortical medullary junction of the frontal lobe. He was found to have hypocalcemia, hyperphosphatemia, and low serum PTH levels, but an increased urinary calcium/creatinine ratio (Table 1). Treatment with calcitriol and calcium was started, and at 7 4/12 yr, nephrocalcinosis and urolithiasis were diagnosed. The patient had an episode of renal colic and passed a calcium oxalate stone. He continues to have muscle cramps, especially in his neck, and headaches. The third son (III-3) presented at 4 2/12 yr with an 18-month history of exercise-induced laryngeal stridor, like his brother (III-2). Laboratory findings showed hypocalcemia, hyperphosphatemia, low serum PTH, and a relatively increased urinary calcium/creatinine ratio (Table 1). The patient was started on calcium and vitamin D supplementation, but immediately after diagnosis the patient had several episodes of severe flank pain. Ultrasound and computed tomography revealed left hydroureteronephrosis and a very small (3-mm) renal stone in the left lower calyceal region. He was changed from calcium carbonate to calcium citrate replacement and has had no further episodes of renal colic.

View larger version (8K): [in this window] [in a new window]   FIG. 1. Pedigree of family K with ADH. Clinical status is indicated by open symbols (unaffected) and solid symbols (ADH). Individuals with normal results on biochemical assessment are indicated by a quartered symbol. The proband is indicated by the arrow.

Leukocyte DNA was isolated using standard methods. Nine primer pairs were used to amplify exons 2–7 (which encode the receptor protein) of the CASR gene, as described previously (3, 8). Forward and reverse primers were modified at their 5' ends by the addition of a T7 or T3 promoter sequence, respectively, to aid in subsequent nucleotide sequencing of the PCR product. After gel purification, PCR products were directly sequenced. For all family members, CASR exon VII was amplified and digested with EarI to test for the presence of the mutation.

Subcloning of CASR exon VII and restriction enzyme analysis

A portion of CASR exon VII was amplified from leukocyte DNA of individuals II-1, II-2, and III-3 and subcloned into the TA cloning vector PCR2.1Topo. Many subclones from each individual were examined by EarI restriction enzyme digestion and scored as either wild type or mutant sequence.

Denaturing HPLC (DHPLC)

The CASR exon VII amplicons from leukocyte DNA of individuals II-1, II-2, III-1, III-2, and III-3 were subjected to DHPLC analysis according to the method described by Oefner and Underhill (14). PCR products were denatured at 95 C for 5 min and cooled to 65 C in 1 C/min decrements. Optimal DNA melting temperatures for specific PCR products were calculated with the DHPLC Melt program (http://insertion.stanford.edu/melt.html). Heteroduplexes were resolved using a Varian ProStar Helix System. Samples of 5 µl crude PCR product were injected and eluted at the optimal temperature by an acetonitrile gradient at a constant flow rate of 0.45 ml/min. The gradient was generated by mixing buffer A [0.1 M triethylamine acetate buffer (pH 7.0), and 0.1 mM EDTA] and buffer B (0.1 M triethylamine acetate buffer (pH 7.0), 25% acetonitrile, and 0.1 mM EDTA]. The gradient was made with linear increments of buffer B from 50–68% over 5.5 min, and the elution profile was monitored by UV spectrometry.

Haplotyping

Genotypes were defined using 10 flanking variable number of tandem repeats markers and 4 intergenic and 1 intragenic single nucleotide polymorphism covering a 9-cM region on chromosome 3.

DNA fingerprint analysis

Genomic DNA samples from individuals II-1, II-2, III-1, III-2, and III-3 were tested using eight microsatellite markers (D5S818, vWF, D13S317, TH01, D7S820, TPOX, D16, and CSFIPO) and the AMELX/Y sex differentiation marker.

MAPK assay

A trans-reporting system (Stratagene, La Jolla, CA) was used to measure the activity of Elk-1, an ETS domain transcription factor targeted by MAPK pathways. For transient transfection, human embryonic kidney (HEK293) cells were trypsinized, plated in six-well dishes in DMEM plus 10% PBS (1–4 x 10⁵ cells/well), and incubated overnight. The next day, cells were transfected with 30 µg/well Superfect reagent with 0.5 µg CASR wild type or mutant or with empty vector (pCDNA3.1), and 0.5 µg each of the Elk-1 reporter constructs (pFA2-Elk-1 and pFR-Luc) and 0.5 µg pCH110 DNA. The following day cells were serum-starved in DMEM containing 0.5 mM CaCl₂ for 8 h and cultured in various concentrations of CaCl₂ ranging from 0.25–15 mM for 16 h. The cells were washed in PBS and lysed in 250 µl lysis buffer (1% Triton X-100, 15 mM MgSO₄, 4 mM EGTA, 1 mM dithiothreitol, and 25 mM glycyl glycine) on ice. Luciferase activity was measured in an EG&G Berthold luminometer (Nashua, NH) using 45 µl cell lysate and D-luciferin. Luciferase activity was normalized to ß-galactosidase. All CASR constructs were derived from a CASR cDNA in which a c-Myc epitope tag was inserted between amino acids 22 and 23 just after the signal peptide cleavage site at amino acid 19 in the CASR NH₂-terminal region (8, 15). We have demonstrated previously that the c-Myc-tagged CASR and untagged CASR behave identically with respect to expression and cell signaling (8, 15).

	Results

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Mutations in CASR assessed by direct sequence analysis

Direct sequence analysis of PCR-amplified CASR exons in the proband (III-2) identified a heterozygous mutation TTCCTC changing codon 788 from phenylalanine to leucine in transmembrane region 5 encoded by exon VII (Fig. 2). This created a new EarI site in the CASR exon VII amplicon, and by restriction enzyme analysis individuals II-1, II-2, and III-1 were found to be normal, and individuals III-2 and III-3 heterozygous, for the mutation (Fig. 2). The exon VII amplicons were directly sequenced in individuals II-1 and II-2, yielding normal sequence.

View larger version (37K): [in this window] [in a new window]   FIG. 2. Detection of a mutation in the CASR gene. A, Direct sequence analysis of the exon 7 genomic DNA amplicon of proband III-2 (left) revealed a heterozygous missense mutation compared with an unrelated normal individual (right). B, Wild-type and mutant sequences of part of exon 7. The restriction enzyme EarI recognition site created by the mutation (TTCCTC, codon 788) is underlined, and the cleavage site is indicated between the first and second nucleotides of codon 790. The new EarI site in the mutant and the common EarI site are shown on the restriction map of the exon 7 amplicon. C, Gel electrophoretic separation of EarI restriction digests of the exon 7 PCR product in all family members (pedigree numbers as in Fig. 1) confirmed the presence of the mutation in individuals III-2 and III-3. Lane M, DNA markers with sizes to the left of the gel. Undigested and EarI-digested exon 7 amplicon sizes are shown to the right.

Molecular analysis using eight microsatellite markers indicated that the family relationships are as stated with a probability of more than 99.9%.

Subcloning of CASR exon VII and restriction enzyme analysis

Subclones of the leukocyte DNA CASR exon VII amplicon inserted into a TA cloning vector were distinguished by EarI restriction enzyme analysis (Fig. 3, A and B). The wild-type/mutant ratios found were: II-1, 123:0 (100% wild type:0% mutant); II-2, 206:20 (91% wild type:9% mutant); and III-3, 26:28 (48% wild type:52% mutant; Fig. 3C). Therefore, the father (II-1) was confirmed as normal, and the son (III-3) as being heterozygous for the mutation. The mother (II-2) was demonstrated to be mosaic for the mutation.

View larger version (25K): [in this window] [in a new window]   FIG. 3. Detection of mosaicism for the CASR mutation in individual II-2 by analysis of subcloned PCR amplicons. A, DNA fragment patterns of EarI-digested recombinant plasmids representing either wild-type or mutant CASR exon VII cloned into the PCR2.1-TOPO vector in either sense or antisense orientation. The sizes of fragments (in base pairs) derived from the insert are shown to the left, and those derived from the vector are shown to the right. B, EarI (E) restriction map of the PCR2.1-TOPO vector and the four alternative inserts. C, Percentage of clones obtained (WT, wild-type; MUT, mutant) after subcloning the CASR exon VII amplicon from leukocyte DNA of individuals II-1 (123 clones examined), II-2 (226 clones examined), and III-3 (54 clones examined).

The CASR exon VII amplicons from leukocyte DNA of all family members were subjected to DHPLC (Fig. 4). The chromatograms for an unrelated normal subject (Fig. 4A), father II-1 (Fig. 4B), and son III-1 (Fig. 4C) only showed homo-duplex formation consistent with a wild-type genotype. The chromatograms for son III-2 (Fig. 4D) and son III-3 (Fig. 4E) showed evidence of homo- and heteroduplex formation consistent with the heterozygous F788L (TTCCTC) mutation. The chromatogram of the amplicon from the mother II-2 shows evidence of approximately 10% of the mutant allele (Fig. 4F).

View larger version (21K): [in this window] [in a new window]   FIG. 4. Detection of mosaicism for the CASR mutation in individual II-2 by DHPLC. Chromatograms of the CASR exon VII amplicon from leukocyte DNA of an unrelated normal individual (A), the father (II-1; B), a son (III-1; C), a son (III-2; D), a son (III-3; E), and the mother (II-2; F).

Of the 15 markers used, eight were informative, and these are the ones shown in Fig. 5. One paternal allele was inherited by the normal son (III-1), and the other paternal allele was inherited by affected sons III-2 and III-3. Therefore, by this analysis alone, paternal involvement in the CASR mutation could not be excluded. There are two possible maternal chromosomal arrangements (one is shown in Fig. 5), and at present (without additional information derived from other family members who are not available to us) the correct arrangement is not known. Recombination of the maternal allele, on which the mutation arose, occurred between marker D3S1303 and the intragenic marker, CASR4I. In the particular scheme shown, son III-1 has inherited the normal maternal allele, and son III-3 has inherited the mutated maternal allele. Son III-2 has inherited the recombinant allele (Fig. 5).

View larger version (22K): [in this window] [in a new window]   FIG. 5. Haplotype analysis in family K. The genotypes for markers on chromosome 3q11.2–24 were determined according to their positions on chromosome 3q, from centromere to telomere. , The chromosome of II-2 that carries the CASR mutation (indicated by the arrow) in a small (10%) proportion of her leukocytes. Individual III-2 has inherited his mother’s normal chromosome 3q 5' to the CASR, but carries the CASR F788L mutation due to recombination.

The ability of the mutant receptor to respond to extracellular calcium relative to the wild-type receptor was examined using a trans-reporting system that measures the activity of Elk-1, an ETS domain transcription factor targeted by MAPK pathways. The wild-type CASR cDNA, when transiently expressed in HEK293 cells, showed a half-maximal response (EC₅₀) of 4.7 ± 0.1 mM (mean ± SE; Fig. 6). The F788L mutant showed a significant leftward shift in its dose-response curve relative to the wild type, with an EC₅₀ of 3.0 ± 0.1 mM (Fig. 6A). This was identical to the response of an activating CASR mutant (A835T; data not shown) that we have characterized previously with respect to its enhanced ability to stimulate intracellular calcium transients (8). This demonstrated the usefulness of the MAPK assay and showed that the F788L mutation was indeed activating. In addition, the previously described F788C mutant (16) was similarly more responsive to extracellular calcium in this assay (Fig. 6A). When equal amounts of wild-type and F788L mutant CASR cDNAs were transiently expressed, the dose-response curve was leftward shifted to a position intermediate between that of the F788L mutant alone and wild-type CASR alone, with an EC₅₀ of 3.9 ± 0.1 mM (Fig. 6B).

View larger version (14K): [in this window] [in a new window]   FIG. 6. Extracellular calcium-evoked increases in MAPK activity in HEK293 cells transiently transfected with either wild-type (WT) or mutant CASR cDNAs (F788L and F788C) or vector pCDNA3.1 (negative control) and a MAPK trans-reporting system. A, Comparison of WT, F788L, and F788C. B, Comparison of WT and F788L, either alone or together. Values shown are the mean ± SEM of four estimations.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

The CASR signals through multiple pathways (see Ref. 2 for review). Initial studies of the functionality of CASR mutants monitored increases in intracellular calcium or inositol 1,4,5-trisphosphate production in response to increases in extracellular calcium. However, the relevance of this pathway to coupling of CASR activation to inhibition of PTH secretion, for example, is not clear. More recently, it has been appreciated that regulation of MAPK by the CASR (predominantly occurring downstream of PKC activation) is of more relevance with respect to parathyroid secretory function (18, 19). In the present study we used a MAPK assay (10) and showed that the F788L mutant was more responsive to increases in extracellular calcium than the wild type. A previous report documented an F788C mutation in the CASR in a Japanese family with familial hypoparathyroidism (16). This involves the same codon as in the present case, but mutated to a different amino acid. The F788C mutant demonstrated a leftward shift in the concentration-response curve with respect to extracellular calcium changes and intracellular calcium increases, similar to what was observed with the F788L mutant in the MAPK assay in the present study. Thus, mutation of phenylalanine 788 to either leucine or cysteine has a similar effect.

Identification of CASR mutations plays an important role in the clinical diagnosis and management of inherited hypocalcemic and hypercalcemic disorders. At present, CASR mutational analysis is conducted by amplifying all six protein-coding exons and flanking intronic sequences from patient leukocyte DNA. This generates a series of approximately 10 amplicons that are then subjected to direct DNA sequencing. It is clear that a more efficient program would be desirable, and we have begun to evaluate DHPLC (20) as a way to screen out the amplicons of wild-type sequence and select only those showing evidence of sequence variation for nucleotide sequencing (unpublished data). In the present study DHPLC was important in confirming that the older brother had a normal genotype, whereas the index case and his younger brother were both heterozygous. It also showed that the father II-1 was of normal genotype, but suggested that about 10% of the mother’s leukocyte DNA bore the F788L mutation. This was confirmed by a more laborious approach, namely, that of restriction enzyme analysis of many subclones of the exon 7 amplicons from key family members, as others have described for tuberous sclerosis (21). Haplotyping would have pointed to the parent of origin if the segregation of parental chromosomes had not been the same for the two affected siblings, but DHPLC provided unambiguous evidence for a maternal origin. It is worth noting that the maternal origin of the mutation prevents direct assessment of germline mosaicism, but acts to confirm the maternal meiotic recombination event suggested by the haplotype analysis.

Mosaicism has important consequences for the clinical assessment of patients, for the interpretation of molecular diagnostic tests, and, most importantly, for genetic counseling (22, 23, 24). In cases like this, the mutation occurs at an early developmental stage and is therefore present in both somatic and gonadal germ cells. Transmission to the offspring is therefore in its complete form (25). The frequency of mosaic mutations can be underestimated, because they often remain undetected during routine mutation analysis, and there may not be subsequent affected siblings to suggest an alternative to a de novo mutation in the affected index case. Given the relatively high frequency of de novo mutations described for familial hypocalciuric hypercalcemia/neonatal severe hyperparathyroidism or ADH (7, 12) and the mosaicism rate for other autosomal disorders in the range of 10–20% (26, 27), it would be anticipated that mosaicism is probably present in a significant number of the de novo familial hypocalciuric hypercalcemia/neonatal severe hyperparathyroidism and ADH cases reported to date. Thus, we would recommend that careful screening of parents be undertaken to uncover low level mosaicism in de novo cases of CASR mutation. At a minimum, parents should be counseled that there is a significant risk (up to 10%) of recurrence in subsequent offspring until such studies can be carried out.

In summary, we present the first report of mosaicism of a CASR mutation. This has important implications for genetic counseling of families with apparently sporadic hypoparathyroidism.

	Acknowledgments

 
We thank all family members for their participation; Xiaoling Wang, Zhanqin Liu, and Betty Wong for technical assistance; Drs. Jim E. Garrett and Kimberley V. Rogers for providing plasmid pHuPCaR4.0 and HEK293 cells; and Dr. Lilia D’Souza-Li for preparing the c-Myc-tagged CASR cDNA expression vector. Electronic-Database Information: The URLs for data in this article are as follows: Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm. nih.gov/Omim/ [for ADH (MIM:60119)]. Calcium-sensing receptor mutation database, http://www.casrdb.mcgill.ca

	Footnotes

 
This work was supported in part by the Canadian Institutes of Health Research (Grant MOP-57730), the Kidney Foundation of Canada (to G.N.H.), and the Natural Sciences and Engineering Research Council and Dairy Farmers of Canada (to D.E.C.C.). We also acknowledge doctoral fellowships from the Canadian Institutes of Health Research and the NCI of Canada (to L.C.) and a studentship from The Royal Victoria Hospital Research Institute (to S.P.).

Abbreviations: ADH, Autosomal dominant hypocalcemia; CASR, calcium-sensing receptor; DHPLC, denaturing HPLC; EC₅₀, half-maximal response.

Received March 10, 2003.

Accepted April 30, 2003.

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Top Abstract Introduction Subjects and Methods Results Discussion References

 

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