Two novel mutations of the calcium-sensing receptor gene affecting the same amino acid position lead to opposite phenotypes and reveal the importance of p.N802 on receptor activity.

OBJECTIVE: Gain-of-function mutations of the calcium-sensing receptor (CASR) gene have been identified in patients with sporadic or familial autosomal dominant hypocalcemia (ADH). Inactivating mutations of the CASR gene cause familial hypocalciuric hypercalcemia (FHH). Here, we report two novel CASR mutations affecting the same amino acid (p.N802); one causes ADH and the other atypical FHH. PATIENTS AND METHODS: The first patient, an 11-year-old girl suffering from hypocalcemia, developed nephrocalcinosis when she was only 5 years old. The second patient is a 30-year-old woman who presented with mild hypercalcemia. PCR amplification of CASR coding exons and direct sequencing of PCR products were used to identify mutations. Site-directed mutagenesis was used to generate mutated CASR cDNAs in an expression plasmid. Using the MAPK assay system and transient transfection of Cos-7 cells with wild-type (WT) and mutated CASR, we studied the responses of these mutated receptors to extracellular Ca(2+) and to the negative allosteric CASR modulator, NPS2143. RESULTS: Two heterozygous missense mutations (p.N802I and p.N802S) affecting a residue in the sixth transmembrane domain of CASR were identified. In functional tests, the response of the p.N802S mutant to calcium was typical of an inactivating mutation. However, the p.N802I mutant had 70% of the maximally stimulated WT receptor activity even in the absence of extracellular calcium. This constitutive activity was only partially inhibited by the inhibitor, NPS2143. CONCLUSIONS: The asparagine at amino acid position 802 appears to be essential for the activity of the CASR protein and is implicated in the mechanism of CASR signaling.

Mild forms of hypophosphatasia mostly result from dominant negative effect of severe alleles or from compound heterozygosity for severe and moderate alleles.

BACKGROUND: Mild hypophosphatasia (HPP) phenotype may result from ALPL gene mutations exhibiting residual alkaline phosphatase activity or from severe heterozygous mutations exhibiting a dominant negative effect. In order to determine the cause of our failure to detect a second mutation by sequencing in patients with mild HPP and carrying on a single heterozygous mutation, we tested the possible dominant effect of 35 mutations carried by these patients. METHODS: We tested the mutations by site-directed mutagenesis. We also genotyped 8 exonic and intronic ALPL gene polymorphisms in the patients and in a control group in order to detect the possible existence of a recurrent intronic mild mutation. RESULTS: We found that most of the tested mutations exhibit a dominant negative effect that may account for the mild HPP phenotype, and that for at least some of the patients, a second mutation in linkage disequilibrium with a particular haplotype could not be ruled out. CONCLUSION: Mild HPP results in part from compound heterozygosity for severe and moderate mutations, but also in a large part from heterozygous mutations with a dominant negative effect.

Improved agarose gel assay for quantification of growth factor-induced cell motility.

Cell chemotaxis is frequently required in normal or pathological situations such as invasion, metastasis, and tumor angiogenesis and may involve many different cell types. At present, no device can simultaneously (i) make morphological observations, (ii) quantify cell migration, (iii) test multiple chemoattracting gradients, and (iv) analyze cell-cell interactions. We developed an agarose-based assay to address these questions. Two glass molds were designed, around which agarose gel could be poured to form specific well shapes. Using a vital nuclear stain (Hoechst 33258), we characterized the migration profile of adherent or suspension cells. Cells could be observed during the entire migration process. We were able to follow cells moving toward chemoattractants or being repulsed by other molecules, and we could estimate average migration speed. Using this inexpensive assay, we were able to obtain precise, reproducible results concerning the chemotactic behavior of different cell types. The resulting data differentiated between chemokinetic and chemotactic movement. Chemotactic potencies could be compared using different criteria, such as the number of attracted cells, induced speed, and morphological aspect. This improved agarose assay appears to be a reliable and inexpensive alternative to other available chemotaxis study tools.

Delayed transport of tissue-nonspecific alkaline phosphatase with missense mutations causing hypophosphatasia.

Hypophosphatasia is a rare genetic disease characterized by diminished bone and tooth mineralization due to deficient activity of tissue-nonspecific alkaline phosphatase (TNSALP). The disease is clinically heterogeneous due to different mutations in the TNSALP gene. In order to determine whether mutated TNSALP proteins may be sequestered, degraded, or subjected to delay in their transport to the cell membrane, we built a plasmid expressing a YFP-TNSALP fluorescent fusion protein allowing the observation of cellular localization in live cells by fluorescence confocal microscopy at different time points after transfection. We studied five mutants (c. 571G>A, c. 653T>C, c. 746G>T, c. 1363G>A and c. 1468A>T) exhibiting various levels of in vitro residual enzymatic activity. While the wild-type protein reached the membrane within the first 24h after transfection, the mutants reached the membrane with delays of 24, 48 or 72 h. For all of the tested mutations, accumulation of the mutated proteins, mainly in the Golgi apparatus, was observed. We concluded that reduced ALP activity of these TNSALP mutants results from structural disturbances and delay in membrane anchoring, and not from compromised catalytic activity.