Abstract
The human calcium-sensing receptor (CASR) is the key controller of extracellular Cao2+ homeostasis, and different mutations in the CASR gene have been linked to different calcium diseases, such as familial hypocalciuric hypercalcemia, severe hyperparathyroidism, autosomal-dominant hypocalcemia (ADH), and Bartter’s syndrome type V. In this study, two generations of a family with biochemically and clinically confirmed ADH who suffered severe muscle pain, arthralgia, tetany, abdominal pain, and fatigue were evaluated for mutations in the CASR gene. The study comprises genotyping of all family members, functional characterization of a potential mutant receptor by in vitro analysis related to the wild-type receptor to reveal an association between the genotype and phenotype in the affected family members. The in vitro analysis of functional characteristics includes measurements of inositol trisphosphate accumulation, Ca2+ mobilization in response to [Ca2+]o-stimulation and receptor expression. The results reveal a significant leftward shift of inositol trisphosphate accumulation as a result of the “gain-of-function” mutant receptor and surprisingly a normalization of the response in (Ca2+)i release in the downstream pathway and additionally the maximal response of (Ca2+)i release was significantly decreased compared to the wild type. However, no gross differences were seen in D126V and the D126V/WT CASR dimeric >250 kDa band expression compared to the WT receptor, however, the D126V and D126V/WT CASR immature ~140 kDa species appear to have reduced expression compared to the WT receptor. In conclusion, in this study, a family with a clinical diagnosis of ADH in two generations was evaluated to identify a mutation in the CASR gene and reveal an association between genotype and phenotype in the affected family members. The clinical condition was caused by a novel, activating, missense mutation (D126V) in the CASR gene and the in vitro functional characteristics of the mutation co-segregated with their individual phenotype.
Highlights
In humans, extracellular calcium concentrations ((Ca2+)o) play a key role in most vital physiological processes, and a tight control of (Ca2+)o is critically important
We report here a novel “gain-of-function” mutation of the calcium-sensing receptor (CASR) that co-segregated with the individual phenotype in two generations diagnosed autosomal-dominant hypocalcemia (ADH)
DNA was purified from peripheral blood and the sequences of the UTR exon 1A/B and the protein-coding exons 2–7 of the CASR gene, including the related boundaries, were analyzed after designing primers for all 9 exons used for PCR amplification, in addition to direct sequence analysis of the PCR amplicons
Summary
Extracellular calcium concentrations ((Ca2+)o) play a key role in most vital physiological processes, and a tight control of (Ca2+)o is critically important. The G protein-coupled calcium-sensing receptor (CASR) plays a major role in systemic calcium homeostasis. The most important physiological function of CASR is to maintain and regulate (Ca2+)o. CASR is functionally expressed in the bone, kidney, and gut, the three major organs involved in calcium homeostasis. (Ca2+)o has key roles in bone biology, in the regulation of different hormones, and in various cellular functions. CASR plays a pivotal role in renal calcium reabsorption and excretion [7], whereas it regulates calcium absorption in the intestine [8, 9]. Hypocalcemia will induce increased excitability of the nervous system with a risk of tetany, which occurs when (Ca2+)o is reduced by 0.40 mmol/L below baseline [15]
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