Identification and functional characterisation of calcium sensing receptor variants in patients presenting with calcium disorders: a thirty-year screening program — ASN Events
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  2. CaSR-5 Session-5: Human genomics of calcium-sending disorders
  3. Identification and functional characterisation of calcium sensing receptor variants in patients presenting with calcium disorders: a thirty-year screening program

Identification and functional characterisation of calcium sensing receptor variants in patients presenting with calcium disorders: a thirty-year screening program (#183)

Bryan K Ward 1 2 , Bronwyn GA Stuckey 1 3 4 , John P Walsh 1 4 , Donald H Gutteridge 1 , Suzanne J Brown 1 , Aaron L Magno 1 2 , Benjamin H Mullin 1 5 , Kassandra M Leatherbarrow 1 , Arthur D Conigrave 6 7 , Scott G Wilson 1 5 8 , Thomas Ratajczak 1 2
  1. Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Nedlands, WA, Australia
  2. Harry Perkins Institute of Medical Research, Centre for Medical Research, University of Western Australia, Nedlands, WA, Australia
  3. Keogh Institute of Medical Research, Nedlands, WA, Australia
  4. Medical School, University of Western Australia, Nedlands, WA, Australia
  5. School of Biomedical Sciences, University of Western Australia, Nedlands, WA, Australia
  6. School of Life and Environmental Sciences, Charles Perkins Centre (D17), University of Sydney, Sydney, NSW, Australia
  7. Department of Endocrinology, Royal Prince Alfred Hospital, Camperdown, NSW, Australia
  8. Department of Twin Research and Genetic Epidemiology, King's College London, London SE1 7EH, United Kingdom

Familial hypocalciuric hypercalcaemia type 1 (FHH-1) and neonatal severe hyperparathyroidism (NSHPT) are caused by heterozygously or homozygously inherited inactivating variants of the calcium sensing receptor gene (CASR), respectively, whereas autosomal dominant hypocalcaemia type 1 (ADH-1) is caused by heterozygously inherited activating variants of the CASR (1). Genetic screening of patients with hypercalcaemia/hypocalcaemia is important to exclude other causes of calcium imbalance to ensure appropriate therapies are applied. CASR variants can occur anywhere in the coding region of the CASR and the identification of a variant alone is not considered sufficient to assign causality; functional characterisation of variants is considered essential (2). We aimed to identify CASR variants in patients/families with calcium disorders, and to functionally characterise novel variants (or those not previously assessed) to confidently assign causality. Sanger sequencing of patient DNA identified variants which were subsequently generated in vitro by site-directed-mutagenesis in a mammalian expression vector. Expression of each variant in HEK293 cells was assessed by Western blot analysis and activity measured following calcium dosing experiments using an IP-One assay. We screened a total of 171 probands suspected of having either FHH or ADH. A total of 26 CASR variants associated with FHH-like biochemistry covering 37 probands, and 6 CASR variants associated with ADH-like biochemistry covering 7 probands were recorded. Of these 32 CASR variants, we have functionally characterised 15. We discuss a number of these including: 1) two inactivating variants that have a dominant negative effect on wild-type receptor, 2) two inactivating variants that fail to produce mature receptor, 3) a variant that is constitutively activating. We conclude that functional assessment of CASR variants from our screening program confirmed the diagnosis of FHH-1 or ADH-1 in 23 probands/families provisionally diagnosed with these disorders.

References:     (1) Gorvin. J Mol Endocrinol. 2019;63(2):R1-R16

                          (2) Richards et al. Genet Medicine. 2015;17(5):405-424