A calcium-sensing receptor allelic series and underdiagnosis of genetically driven hypocalcemia

Abstract

The availability of genomic sequencing has revealed that variants in genes that cause rare monogenic disorders are relatively common, which raises the question of variant pathogenicity. Autosomal-dominant hypocalcemia type 1 (ADH1) is a rare genetic form of hypoparathyroidism caused by gain-of-function (GoF) variants in the calcium-sensing receptor (CaSR) encoded by CASR. We examined the prevalence, penetrance, and expressivity of GoF CASR variants in the UK Biobank (UKB; n = 433,793), All of Us (AOU; n = 229,987), and Mass General Brigham Biobank (n = 39,081). Individuals with previously reported ADH1-associated variants indeed showed ADH1 symptoms, including hypocalcemia (60% in the UKB and 78% in AOU). However, less than half had an ADH1-relevant diagnosis code (17% in the UKB and 44% in AOU), suggesting that individuals with ADH1 are present in these biobanks but may be underdiagnosed. We then developed a scoring algorithm and identified nine low-frequency ADH1-associated variants, which were further validated using genetic sequencing of individuals with nonsurgical hypoparathyroidism (n = 169) and an in vitro functional assay. These nine variants have an intermediate effect and frequency relative to previously reported ADH1-associated variants, completing an allelic series with respect to serum calcium, and alone are responsible for a symptom burden roughly equivalent to all previously reported ADH1-associated variants. Our work indicates that hypocalcemia due to GoF in CASR with ADH1-associated symptoms is underdiagnosed, provides a deeper understanding of the genotype-phenotype relationship of CASR variants, and illustrates that variants in genes underlying rare disorders may cause a much greater symptom burden than currently appreciated.

Keywords: ADH1; Mendelian disorder; autosomal-dominant hypocalcemia type 1; hypocalcemia; hypoparathyroidism; incomplete penetrance; monogenic disorder; rare disease; symptom burden; variable expressivity.

Graphical abstract

Figure 1

A scoring method to identify variants associated with ADH1 (A) The ADH1 score for each variant and each self-reported ethnicity in the UKB or genetically determined ancestry in AOU was calculated as the weighted sum of sub-scores corresponding to strength of association with ADH1 phenotypes and other variant characteristics. Variant location had a positive sub-score when variants were within known hotspots of GoF variants (aa positions 116–136 and 819–837). SOC treatment, standard-of-care treatment, e.g., calcium supplementation or parathyroid hormone. (B) Comparison of synonymous variants (bars) and previously described ADH1-associated variants (red arrows) shows separation of their scores. (C) Distribution of all scored variants (missense, frameshift, or nonsense). (D) Summary of biobank scoring analysis. (E) Comparison of ADH1 scores of variants found in both the UKB and AOU. Highlighted region indicates variants that scored higher than the 1.5 threshold in both the UKB and AOU. (F) Boxplot comparing effects on serum calcium, estimated in the MGB Biobank, of variants that scored above/below the threshold of 1.5 in AOU and the UKB. The boxes represent the interquartile range (IQR), the whiskers extend to 1.5 times the IQR, and the dots are outside of this range. The dashed line indicates the mean. (G) Bar chart showing the symptom burden of ADH1 (per 100,000 individuals in the UKB) from known variants, additional variants, and the combined total. ADH1, autosomal dominant hypocalcemia type 1; AOU, All of Us; a.u., arbitrary units; MGB, Mass General Brigham; SOC, standard of care; UKB, UK Biobank.

Figure 2

In vitro functional assay to characterize variant sensitivity to extracellular calcium HEK293 cells expressing each CASR variant were exposed to various concentrations of extracellular calcium, and calcium signaling activity was determined using Fluo-8 AM. c.220A>C (p.Met74Leu) was a negative control, and c.310G>A (p.Val104Ile) was a positive control. Each experiment was run 5 times. ^∗p < 0.05 and ^∗∗p < 0.01. p values are not reported for tests that are not statistically significant or near significance.

Figure 3

Genetic architecture of serum calcium with respect to CASR variation Boxplot of calcium effects for sets of variants. The boxes represent the interquartile range (IQR), the whiskers extend to 1.5 times the IQR, and the dots are outside of this range. Additional variants are those that scored >1.5 in both the UKB and AOU (as presented in Tables 3 and S10). The ADH1 score >1.5 categories include the additional variants as well as all others that met the score criteria for each biobank. ADH1, autosomal dominant hypocalcemia type 1; AOU, All of Us; IQR, interquartile range; UKB, UK Biobank.