J Pediatr Endocr Met 2014; 27(9-10): 851–856
Alicia Diaz-Thomas*, John Cannon, Pallavi Iyer, Almundher Al-Maawali, Mohammed Fazalullah, Frank Diamond, O. Thomas Mueller, Allen W. Root and Saif Alyaarubi
A novel CASR mutation associated with neonatal severe hyperparathyroidism transmitted as an autosomal recessive disorder Abstract Background: Neonatal severe primary hyperparathyroidism (NSHPT, MIM 239200) is most often an isolated disorder that is due to biallelic inactivating mutations in the CASR, the gene encoding the calcium sensing receptor; NSHPT is inherited from parents with familial hypocalciuric hypercalcemia, each of whom has one mutated CASR allele. Objectives: To report clinical and genetic findings in a brother and sister with NSHPT due to a novel mutation in the CASR transmitted as an autosomal recessive trait and to examine the functional effect of the mutation. Subjects and methods: A brother and sister with marked hypercalcemia due to NSHPT were identified; the boy also had craniosynostosis requiring surgical repair. The genotyping of the CASR in both children and their parents who were eucalcemic and normophosphatemic was undertaken. In order to examine the significance of the variant CASR identified, the CASR variant was expressed in vitro and examined by three computer computational programs [PolyPhen2, MutationTaster, Sorting Intolerant From Tolerant (SIFT)] designed to evaluate the effect of a nucleotide variant on the structure and likely functional consequence upon the protein product. Results: A sequence variant in the CASR was identified [G > T point mutation at nucleotide c.2303 in exon 7 (c.2303G > T) resulting in the replacement of glycine by
*Corresponding author: Alicia Diaz-Thomas, MD, University of Tennessee Health Science Center, Le Bonheur Children’s Hospital, Memphis, TN, 50 N Dunlap St, CFRT, Ste 511, Memphis, TN 38103, Phone: 901.287.6221, Alt. Phone: 504.723.2365, E-mail: [email protected], [email protected] John Cannon: USF/ACH Children’s Research Institute, St. Petersburg, FL, USA Pallavi Iyer, Frank Diamond and Allen W. Root: All Children’s Hospital/Johns Hopkins Medicine, St. Petersburg, FL, USA Almundher Al-Maawali, Mohammed Fazalullah and Saif Alyaarubi: Sultan Qaboos University Hospital, Oman O. Thomas Mueller: ACH Molecular Genetics Laboratory, St. Petersburg, FL, USA
valine at codon 768 (p.Gly768Val)]. Two copies of this CASR variant were present in the genome of the siblings while a single copy of the CASR variant was present in both of the clinically and biochemically normal parents, a pattern of transmission consistent with autosomal recessive inheritance of NSHPT in this family. When expressed in HEK293 cells in vitro, the novel Gly768Val variant did not interfere with protein generation or migration to the cell membrane in vitro. The analysis of the functional effect of the Gly768Val CASR variant by the PolyPhen2, MutationTaster, and Sorting Intolerant From Tolerant computer programs revealed that this mutation was very likely to be deleterious. Conclusion: The NSHPT associated with biallelic Gly768Val mutations of the CASR in two siblings with severe hypercalcemia and hyperparathyroidism and their clinically and biochemically normal heterozygous parents was transmitted as an autosomal recessive disorder in this family. Keywords: calcium sensing receptor (CaSR); familial/ hereditary hypocalciuric hypercalcemia; neonatal hypercalcemia; neonatal severe hyperparathyroidism. DOI 10.1515/jpem-2013-0343 Received August 21, 2013; accepted April 1, 2014; previously published online May 22, 2014
Introduction The calcium sensing receptor (CaSR) is a seven transmembrane G-protein coupled receptor that is essential for the maintenance of normal calcium homeostasis. Its intracellular signal is transmitted through the Gαq and Gα11 subunits of its G-protein. Acting through the CaSR on the cell membrane of the parathyroid chief cell, the rising levels of ionized calcium (Ca2+) normally suppresses the transcription of PTH (encoding parathyroid hormone); in the renal thick ascending limb of the loop of Henle, the stimulation of the CaSR by Ca2+ inhibits the renal tubular reabsorption
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852 Diaz-Thomas et al.: A novel CASR mutation associated with neonatal severe hyperparathyroidism of calcium. Both loss- and gain-of-function mutations in the CASR (chromosome 3q21.1, MIM 601199) have been described (1). Monoallelic inactivating mutations of the CASR result in familial hypocalciuric hypercalcemia type 1 (HHC1 – MIM 145980), a disorder characterized by asymptomatic hypercalcemia and paradoxical hypocalciuria that is transmitted as an autosomal dominant trait. Neonatal severe hyperparathyroidism (NSHPT – MIM 239200) usually occurs in an offspring, both of whose parents have HHC1 and is due to the biallelic loss-offunction CASR mutations. (NSHPT may also develop in an affected neonate bearing only one inactivating variant of the CASR allele if the mother is normal.) Monoallelic activating mutations of the CASR lead to familial isolated (autosomal dominant) hypoparathyroidism type 1 (FIH1, MIM 146200). Additionally, autosomal dominant HHC has been mapped to two other loci – specifically chromosome 19p13.3 (HHC2), and 19q13 (HHC3). The HHC2 (MIM 145981) has been attributed to loss-of-function mutations in the GNA11 encoding guanine nucleotide-binding protein, alpha 11 (MIM 139313), one of the G-protein α subunits through which the CaSR transduces its intracellular signal (2). The gain-of-function mutations in GNA11 have been found as well in patients with FIH type 2 (2, 3). Inactivating mutations in the AP2S1 encoding adaptor protein-2α subunit (MIM 602242) involving solely the Arg15 residue of AP2S1 has been identified in patients with HHC3 (MIM 600740) (4). The AP2S1 is one of the four subunits that make up a protein critical for the clathrin-related endocytosis of G-protein coupled receptors. Inactivating variants of the AP2S1 decrease sensitivity of CaSR to Ca2+, reduce its rate of endocytosis, and impede intracellular signal transduction. Patients with HHC1 have mild to moderate hypercalcemia, inappropriately normal PTH levels, and hypocalciuria. Most patients with HHC1 are asymptomatic and identified incidentally. Neonates with NSHPT fail to thrive; they have marked hypercalcemia (total Ca > 3.75 mmol/L), highly elevated PTH values, and inappropriately low urine calcium excretion. Mutations leading to HHC1, NSHPT, and autosomal dominant hypoparathyroidism have been identified throughout the CASR nucleotide sequence, but most often at those sites encoding the calcium-binding loci of the extracellular domain (1). Interestingly, activating and inactivating mutations in the CASR may occur in the same or adjacent codons. The biologic effect of a specific mutation is determined by the amino acid substitution and its influence upon the three-dimensional conformation of the receptor (5). We describe the clinical and biochemical findings in a brother and sister, offspring of eucalcemic Pakistani
first cousins, with NSHPT, due to a novel homozygous mutation (c.2303G→T; p.Gly768Val) in the CASR transmitted as an autosomal recessive characteristic. The lad also had craniosynostosis requiring surgical repair. We have examined protein expression and movement of the Gly768Val variant in vitro and compared these findings to those observed in a CaSR bearing a gain-of-function mutation (Glu767Gln) in a neighboring codon and wild type CaSR (6). Additionally, the effect of the mutation upon the protein structure and function was evaluated by the PolyPhen2, MutationTaster, and SIFT computer programs (7–9).
Materials and methods This study was approved by the Institutional Review Board of All Children’s Hospital/Johns Hopkins Medicine, St Petersburg, FL, USA. The informed consent for the molecular studies of the siblings was obtained from their parents. The genotyping and molecular studies were performed by standard methods (see Supplemental Materials).
Results Clinical history Patient 1 was a male who was born to a 24-year-old, gravida 1, para 1, mother and a 28-year-old father of Pakistani descent (Figure 1A). The pregnancy was complicated by a fetal intrauterine growth restriction; the vaginal delivery was induced at 36 weeks gestation because of decreased fetal movements. The birth weight was 1.7 kg, birth length 45 cm, and head circumference 29 cm, all of which were less than their respective 10th percentiles. The physical examination of the neonate revealed brachycephaly, downslanted palpebral fissures, and maxillary hypoplasia. The anterior fontanel measured 1.5 × 2 cm; the cranial sutures were prominent. Cranial radiographs and cranial computed tomography demonstrated fusion of the coronal, sphenofrontal, and frontoethmoidal cranial sutures; the skeletal survey revealed osteopenia. A perimembranous ventricular septal defect (VSD) was also present. Further studies revealed (Figure 1B) hypercalcemia, hypophosphatemia, hyperalkaline phosphatasia, and an elevated level of parathyroid hormone (PTH). Serum concentrations of magnesium (1.0 mmol/L), calcitriol (369 pmol/L), and calcidiol (66.9 nmol/L) were normal for age. The spot urinary calcium/creatinine ratio was 0.8 mmol/mol creatinine. For management of hypercalcemia, the neonate
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Diaz-Thomas et al.: A novel CASR mutation associated with neonatal severe hyperparathyroidism 853
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Figure 1 (A) Pedigree of the family reported demonstrating autosomal recessive of NSHPT. (B) Table listing serum analyte concentrations in the reported family.
received furosemide, hydrocortisone, calcitonin, pamidronate, and a low calcium diet with temporary restoration of eucalcemia. At age 4 months because of unremitting hyperparathyroidism, patient 1 underwent subtotal parathyroidectomy. Immediately prior to surgery the physical examination revealed length 53.5 cm and weight 3.7 kg (less than their respective 3rd percentiles), brachycephaly, biparietal bossing, down-slanting palpebral fissures, and a Grade III/VI systolic murmur along the left sternal border. Preoperatively, the 24-h urinary calcium excretion was 0.68 mmol/mmol creatinine. After the surgical excision of 3.5 parathyroid glands, the iPTH levels declined and hypocalcemia developed requiring temporary supplementation with calcium gluconate (Figure 2). Serum calcium values
returned to the hypercalcemic range by the 13th postoperative day and persisted thereafter. The pathologic examination of the parathyroid glands revealed hyperplasia. The combined weight of three hyperplastic parathyroid glands was 160 mg (the normal weight of 4 parathyroid glands in a 3-month-old infant is approximately 25.4 mg). At age 23 months, patient 1 had delayed motor and speech development. He underwent the surgical repair of craniosynostosis at 49 months of age. At age 54 months, patient 1 remained hypercalcemic (2.92 mmol/L). Patient 2, the younger sister of patient 1, was the product of a 38-week uncomplicated pregnancy and vaginal delivery. The birth weight was 3.03 kg, length 50 cm, head circumference 34 cm. The physical examination at
3.375 Patient 1
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Figure 2 Changes in serum concentrations of calcium after subtotal parathyroidectomy in siblings with NSHPT.
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854 Diaz-Thomas et al.: A novel CASR mutation associated with neonatal severe hyperparathyroidism
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Figure 3 Site of the Gly768Val mutation of CASR in the family in which NSHPT was transmitted as an autosomal recessive characteristics.
birth disclosed brachycephaly, prominent cranial sutures, hypertelorism, and down-slanted palpebral fissures. At birth, patient 2 was hypercalcemic and hypophosphatemic and had high serum concentrations of PTH and 1,25-dihydroxyvitamin D (405 pmol/L, RR T point mutation at nucleotide c.2303 in exon 7 (c.2303G > T) resulting in the replacement of glycine by valine at codon 768 (p.Gly768Val = G768V) (Robert Thacker Ph.D., OCDEM, Genetics Laboratories, Headington, Oxford) (Figure 3). Concurrent sequencing of the CASR (GENE DX, Gaithersberg, MD, USA) confirmed the Gly768Val mutation and also revealed a homozygous variation at codon 986 (Ala986Ser), a common polymorphism that has been associated with alterations in circulating calcium levels in healthy adults (10). The homozygosity for this sequence variant has also been identified in a neonate with transient hypercalcemia (11). The genotyping of the CASR from peripheral monocytes of both parents, and
from a paraffin embedded parathyroid gland of patient 2 confirmed the Gly768Val mutation in these four subjects. Both children were homozygous, and both parents were heterozygous for the Gly768Val CASR variant.
Functional effect of mutant CASR The Gly768Val variant is situated in the extracellular loop of the CaSR that connects transmembrane domain 2 to transmembrane domain 3, an otherwise evolutionarily conserved site among mammals (Figure 3). The functional significance of the novel Gly768Val mutation was examined and compared to the wild type CASR and the CASR bearing an adjoining gain-of-function variant, Glu767Gln (6). When the Gly768Val, Glu767Gln, and the wild type CASRS were expressed in transfected HEK293 cells, protein was synthesized and migrated to the cell surface in each transfected cell line (Supplemental Figure 1). The functional significance of the Gly768Val CASR variant was further examined utilizing three computational programs (Polymorphism Phenotyping, version 2 (PolyPhen2) – http://genetics.bwh.harvard.edu/pph2; MutationTaster – http://mutationassessor.org; SIFT – http://sift-dna.org) that estimate the functional effect of a specific change in the amino acid sequence of a protein (7–9). PolyPhen2 evaluates both the amino acid sequence and the structure of the altered protein; PolyPhen2 gauged the Gly768Val variant of CASR to be “probably damaging” with a score of 1.0 (maximum). MutationTaster calculated a Functional Impact score of 3.345 for the Gly768Val variant of the CASR indicating that this mutation is likely to have an adverse effect on protein function of medium significance. The SIFT analysis predicted that the Gly768Val variant was “damaging.” The neighboring gain-of-function variant, Glu767Gln, was likewise assessed using the same three computational programs and also felt to produce an altered protein that would be deleterious to the function of the CaSR receptor.
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Diaz-Thomas et al.: A novel CASR mutation associated with neonatal severe hyperparathyroidism 855
Discussion Human CaSR is a 1078 amino acid (aa) protein containing three distinct regions: a large extracellular domain (aa 1-612), seven transmembrane domains (aa 613-862), and an intracellular tail domain (aa 863-1078) (5). The agonist binding in the cleft of the Venus flytrap (VFT) structure within the extracellular domain induces conformational changes leading to its closure and consequent three-dimensional changes in the transmembrane and tail domains of CaSR that activate intracellular signal transduction primarily through the Gαq and Gα11 subunits of G-protein (2, 12). The Gαq and Gα11 subunits stimulate phospholipase C activity, and the inositol phosphate signal transduction pathway leading to increased intracellular Ca2+ levels, and the activation of the mitogen-activating protein kinase signaling cascade eventuating in the regulation of gene transcription (13). The effects of inactivating mutations in the genes encoding a g-protein coupled receptor (GPCR) can be classified based on the functional consequence of the mutation: class I mutations interfere with receptor biosynthesis, class II mutations disrupt trafficking of the receptor to the cell surface, class III mutations interfere with ligand binding, class IV mutations impede receptor activation, and class V mutations have none of the above characteristics (14). In the majority of patients with familial hypocalciuric hypercalcemia (FHH), the disorder is transmitted as an autosomal dominant (monoallelic) trait likely explained by the dominant negative activity of the mutant CaSR receptor on the wild type CaSR (5). Inasmuch as the parents of the patients described in this report were asymptomatic and eucalcemic heterozygous carriers of the CASR mutation (Gly768Val) while the children bearing the mutation on both alleles (homozygous) were hypercalcemic with marked hyperparathyroidism, we conclude that NSHPT due to the Gly768Val variant of the CASR is transmitted as an autosomal recessive trait. Recently, the Gln459Arg inactivating mutation in the extracellular domain of CASR was found to be transmitted as an autosomal recessive disorder (15). The present study demonstrates that neither the Gly768Val loss-of-function mutation in the CASR nor the adjacent gain-of-function mutation (Glu767Gln) interferes with synthesis of the aberrant CaSR protein or its transit to the cell membrane in transfected human cells. The three computer programs that model the impact of an amino acid variant upon function of the parent protein predict that the Gly768Val mutation of CaSR will have an adverse effect. The biochemical and clinical effects of these mutations most likely lie in their interaction with
Ca2+, or in the manner in which they affect intracellular signaling. Further studies examining the spatial configurations of the mutant CaSRs and the effect of these alterations on dimerization of the variant receptors with the wild type CaSR, the binding affinities of the altered receptors for Ca2+, and their effects on the intracellular signal transduction pathways activated by the interaction of Ca2+ and the CaSR are required to identify the basic biochemical abnormalities these mutations confer upon the CaSR function. However, employing three computer programs that predict the effects of amino acid variations upon the configuration and function of the parent protein demonstrated that the CASR Gly768Val variant is likely to affect adversely the activity of the CaSR protein. Therapeutically, understanding the functional consequences of a mutation in the CaSR permits the identification of the patient who may respond to allosteric modifiers of the CaSR, i.e., a calcimimetic such as cinacalcet or a calcilytic agent. Indeed, a newborn with NSHPT associated with an Arg185Gln mutation in the CASR has been treated with cinacalcet with long-term normalization of the serum calcium level and suppression of serum PTH concentrations (16). Perhaps a calcimimetic agent would be useful in the management of the siblings described in this report. Another observation of interest is the presence of congenital craniosynostosis in the male subject, an abnormality that has not previously been recorded in patients with the CASR mutations. The CASR is expressed in the osteoblasts, osteocytes, and bone marrow stromal cells. The calcium acting through the CaSR inhibits the differentiation of osteoclasts and enhances the activity of osteoblasts (17). The CASR is also expressed in murine calvarial osteoblasts where it regulates their proliferative capacity acting through the Jun N-terminal kinase (JNK) pathway rather than the ERK signaling pathway (18, 19). Perhaps the inactivating mutation of CASR in the calvarial osteoblasts or osteocytes of one of our patients was of pathogenetic significance in the development of craniosynostosis. Further evaluation of the effect of the Gly768Val mutation of the CASR upon intracellular signal transduction pathways would be of interest. We conclude that NSHPT due to the Gly768Val variant of the CASR is transmitted as an autosomal recessive trait. This mutation does not interfere with the generation or movement of the protein product to the cell membrane. Acknowledgments: The authors thank Gary Litman, PhD, for enabling these studies to be conducted in his laboratory; Marci O’Driscoll and Larry Dishaw, PhD, for
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856 Diaz-Thomas et al.: A novel CASR mutation associated with neonatal severe hyperparathyroidism invaluable technical assistance; Dorothy Shulman, MD, E. Verena Jorgensen, MD, and Dewey Eichler, PhD, for helpful discussion.
Conflict of interest statement Authors’conflict of interest disclosure: The authors have no financial or competing interests to disclose. Research funding: The present work was conducted without any extramural funding.
References 1. Hannan FM, Nesbit MA, Zhang C, Cranston T, Curley AJ, et al. Identification of 70 calcium-sensing receptor mutations in hyperand hypo-calcaemic patients: evidence for clustering of extracellular domain mutations at calcium-biding sites. Hum Molec Genet 2012;21:2768–78. 2. Nesbit MA, Hannan FM, Howles SA, Babinsky VN, Head RA, et al. Mutations affecting G-protein subunit α11 in hypercalcemia and hypocalcemia. N Engl J Med 2013;368:2476–86. 3. Mannstadt M, Harris M, Bravenboer B, Chitturi S, Dreijerink KM, et al. Germline mutations affecting Gα11 in hypoparathyroidism. N Engl J Med 2013;368:2532–4. 4. Nesbit MA, Hannan FM, Howles SA, Reed AA, Cranston T, et al. Mutations in AP2S1 cause familial hypocalciuric hypercalcemia type 3. Nature Genet 2013;45:93–7. 5. Hu J, Spiegel AM. Structure and function of the human calcium sensing receptor: insights from natural and engineered mutations and allosteric modulators. J Cell Mol Med 2007;11:908–22. 6. Letz S, Rus R, Haag C, Dörr HG, Dörr H-G, et al. Novel activating mutations of the calcium-sensing receptor: the calcilytic NPS2143 mitigates excessive signal transduction of mutant receptors. J Clin Endocrinol Metab 2010;95:E229–33. 7. Gnad F, Baucom A, Mukhyaia K, Manning G, Zhang Z. Assessment of computational methods for predicting the effects of missense mutations in human cancers. BMC Genomics. 14(suppl 3):S7. DOI:10.1186/1471-2164-14-S3-S7. 8. Reva B, Antipin Y, Sander C. Predicting the functional impact of protein mutations: application to cancer genomics. Nucleic Acids Res 2011 July 3.
9. Kumar P, Henikoff S, Ng PC. Predicting the effects of coding non-synonymous variants on protein function using the SIFT program. Nat Prot 2009;4:1073–81. 10. Kapur K, Johnson T, Beckmann ND, Sehmi J, Tanaka T, et al. Genome-wide meta-analysis for serum calcium identifies significantly associated SNPs near the calcium-sensing receptor (CASR) gene. PLOS Genetics 2010;6:e1001035. DOI: 10.1371/journal.pgen.1001035. 11. Jack MM, Sone ML, Clifton-Bligh R. Neonatal hypercalcemia due to polymorphisms of the calcium sensing receptor. J Pediatr Endocrinol Metab 2009;22:561–3. 12. Hu J, McLarnon SJ, Mora S, Jiang J, Thomas C, et al. A region in the seven-transmembrane domain of the human Ca2+ receptor critical for response to Ca2+. J Biol Chem 2005;280:5113–20. 13. Magno AL, Ward BK, Ratajczak T. The calcium-sensing receptor: a molecular perspective. Endocrine Reviews 2011;32:3–30. 14. White E, McKenna J, Cavanaugh A, Breitwieser GE. Pharmacochaperone-mediated rescue of calcium-sensing receptor loss-of-function mutants. Mol Endocrinol 2009; 23:1115–23. 15. Lietman S, Tenenbaum-Rakover Y, Jap TS, Yi-Chi W, De-Ming Y, et al. A novel loss-of-function mutation, Gln459Arg, of the calcium-sensing receptor gene associated with apparent autosomal recessive inheritance of familial hypocalciuric hypercalcemia. J Clin Endocrinol Metab 2009;94:4372–9. DOI: 10.1210/jc.2008-2484. 16. Reh CM, Hendy GN, Cole DE, Jeandron DD. Neonatal hyperparathyroidism with a heterozygous calcium-sensing receptor (CASR) R185Q mutation: clinical benefit from cinacalcet. J Clin Endocrinol Metab 2011;96:E707–12. 17. Egbuna OI, Brown EM. Hypercalcaemic and hypocalcaemic conditions due to calcium-sensing receptor mutations. Best Pract Res Clin Rheumatol 2008;22:129–48. 18. Brown EM, Lian JB. New insights in bone biology: unmasking skeletal effects of the extracellular calcium-sensing receptor. Science Signaling 2008;35:pe40. DOI: 10.1126/ scisignal.135pe40. 19. Chattopadhyay N, Yano S, Tfelt-Hansen J, Rooney P, Kanuparthi D, et al. Mitogenic action of calcium sensing receptor on rat calvarial osteoblasts. Endocrinology 2004;145:3451–62.
Supplemental Material: The online version of this article (DOI: 10.1515/jpem-2013-0343) offers supplementary material, available to authorized users.
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Alicia Diaz-Thomas*, John Cannon, Pallavi Iyer, Almundher Al-Maawali, Mohammed Fazalullah, Frank Diamond, O. Thomas Mueller, Allen W. Root and Saif Alyaarubi
A novel CASR mutation associated with neonatal severe hyperparathyroidism transmitted as an autosomal recessive disorder Abstract Background: Neonatal severe primary hyperparathyroidism (NSHPT, MIM 239200) is most often an isolated disorder that is due to biallelic inactivating mutations in the CASR, the gene encoding the calcium sensing receptor; NSHPT is inherited from parents with familial hypocalciuric hypercalcemia, each of whom has one mutated CASR allele. Objectives: To report clinical and genetic findings in a brother and sister with NSHPT due to a novel mutation in the CASR transmitted as an autosomal recessive trait and to examine the functional effect of the mutation. Subjects and methods: A brother and sister with marked hypercalcemia due to NSHPT were identified; the boy also had craniosynostosis requiring surgical repair. The genotyping of the CASR in both children and their parents who were eucalcemic and normophosphatemic was undertaken. In order to examine the significance of the variant CASR identified, the CASR variant was expressed in vitro and examined by three computer computational programs [PolyPhen2, MutationTaster, Sorting Intolerant From Tolerant (SIFT)] designed to evaluate the effect of a nucleotide variant on the structure and likely functional consequence upon the protein product. Results: A sequence variant in the CASR was identified [G > T point mutation at nucleotide c.2303 in exon 7 (c.2303G > T) resulting in the replacement of glycine by
*Corresponding author: Alicia Diaz-Thomas, MD, University of Tennessee Health Science Center, Le Bonheur Children’s Hospital, Memphis, TN, 50 N Dunlap St, CFRT, Ste 511, Memphis, TN 38103, Phone: 901.287.6221, Alt. Phone: 504.723.2365, E-mail: [email protected], [email protected] John Cannon: USF/ACH Children’s Research Institute, St. Petersburg, FL, USA Pallavi Iyer, Frank Diamond and Allen W. Root: All Children’s Hospital/Johns Hopkins Medicine, St. Petersburg, FL, USA Almundher Al-Maawali, Mohammed Fazalullah and Saif Alyaarubi: Sultan Qaboos University Hospital, Oman O. Thomas Mueller: ACH Molecular Genetics Laboratory, St. Petersburg, FL, USA
valine at codon 768 (p.Gly768Val)]. Two copies of this CASR variant were present in the genome of the siblings while a single copy of the CASR variant was present in both of the clinically and biochemically normal parents, a pattern of transmission consistent with autosomal recessive inheritance of NSHPT in this family. When expressed in HEK293 cells in vitro, the novel Gly768Val variant did not interfere with protein generation or migration to the cell membrane in vitro. The analysis of the functional effect of the Gly768Val CASR variant by the PolyPhen2, MutationTaster, and Sorting Intolerant From Tolerant computer programs revealed that this mutation was very likely to be deleterious. Conclusion: The NSHPT associated with biallelic Gly768Val mutations of the CASR in two siblings with severe hypercalcemia and hyperparathyroidism and their clinically and biochemically normal heterozygous parents was transmitted as an autosomal recessive disorder in this family. Keywords: calcium sensing receptor (CaSR); familial/ hereditary hypocalciuric hypercalcemia; neonatal hypercalcemia; neonatal severe hyperparathyroidism. DOI 10.1515/jpem-2013-0343 Received August 21, 2013; accepted April 1, 2014; previously published online May 22, 2014
Introduction The calcium sensing receptor (CaSR) is a seven transmembrane G-protein coupled receptor that is essential for the maintenance of normal calcium homeostasis. Its intracellular signal is transmitted through the Gαq and Gα11 subunits of its G-protein. Acting through the CaSR on the cell membrane of the parathyroid chief cell, the rising levels of ionized calcium (Ca2+) normally suppresses the transcription of PTH (encoding parathyroid hormone); in the renal thick ascending limb of the loop of Henle, the stimulation of the CaSR by Ca2+ inhibits the renal tubular reabsorption
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852 Diaz-Thomas et al.: A novel CASR mutation associated with neonatal severe hyperparathyroidism of calcium. Both loss- and gain-of-function mutations in the CASR (chromosome 3q21.1, MIM 601199) have been described (1). Monoallelic inactivating mutations of the CASR result in familial hypocalciuric hypercalcemia type 1 (HHC1 – MIM 145980), a disorder characterized by asymptomatic hypercalcemia and paradoxical hypocalciuria that is transmitted as an autosomal dominant trait. Neonatal severe hyperparathyroidism (NSHPT – MIM 239200) usually occurs in an offspring, both of whose parents have HHC1 and is due to the biallelic loss-offunction CASR mutations. (NSHPT may also develop in an affected neonate bearing only one inactivating variant of the CASR allele if the mother is normal.) Monoallelic activating mutations of the CASR lead to familial isolated (autosomal dominant) hypoparathyroidism type 1 (FIH1, MIM 146200). Additionally, autosomal dominant HHC has been mapped to two other loci – specifically chromosome 19p13.3 (HHC2), and 19q13 (HHC3). The HHC2 (MIM 145981) has been attributed to loss-of-function mutations in the GNA11 encoding guanine nucleotide-binding protein, alpha 11 (MIM 139313), one of the G-protein α subunits through which the CaSR transduces its intracellular signal (2). The gain-of-function mutations in GNA11 have been found as well in patients with FIH type 2 (2, 3). Inactivating mutations in the AP2S1 encoding adaptor protein-2α subunit (MIM 602242) involving solely the Arg15 residue of AP2S1 has been identified in patients with HHC3 (MIM 600740) (4). The AP2S1 is one of the four subunits that make up a protein critical for the clathrin-related endocytosis of G-protein coupled receptors. Inactivating variants of the AP2S1 decrease sensitivity of CaSR to Ca2+, reduce its rate of endocytosis, and impede intracellular signal transduction. Patients with HHC1 have mild to moderate hypercalcemia, inappropriately normal PTH levels, and hypocalciuria. Most patients with HHC1 are asymptomatic and identified incidentally. Neonates with NSHPT fail to thrive; they have marked hypercalcemia (total Ca > 3.75 mmol/L), highly elevated PTH values, and inappropriately low urine calcium excretion. Mutations leading to HHC1, NSHPT, and autosomal dominant hypoparathyroidism have been identified throughout the CASR nucleotide sequence, but most often at those sites encoding the calcium-binding loci of the extracellular domain (1). Interestingly, activating and inactivating mutations in the CASR may occur in the same or adjacent codons. The biologic effect of a specific mutation is determined by the amino acid substitution and its influence upon the three-dimensional conformation of the receptor (5). We describe the clinical and biochemical findings in a brother and sister, offspring of eucalcemic Pakistani
first cousins, with NSHPT, due to a novel homozygous mutation (c.2303G→T; p.Gly768Val) in the CASR transmitted as an autosomal recessive characteristic. The lad also had craniosynostosis requiring surgical repair. We have examined protein expression and movement of the Gly768Val variant in vitro and compared these findings to those observed in a CaSR bearing a gain-of-function mutation (Glu767Gln) in a neighboring codon and wild type CaSR (6). Additionally, the effect of the mutation upon the protein structure and function was evaluated by the PolyPhen2, MutationTaster, and SIFT computer programs (7–9).
Materials and methods This study was approved by the Institutional Review Board of All Children’s Hospital/Johns Hopkins Medicine, St Petersburg, FL, USA. The informed consent for the molecular studies of the siblings was obtained from their parents. The genotyping and molecular studies were performed by standard methods (see Supplemental Materials).
Results Clinical history Patient 1 was a male who was born to a 24-year-old, gravida 1, para 1, mother and a 28-year-old father of Pakistani descent (Figure 1A). The pregnancy was complicated by a fetal intrauterine growth restriction; the vaginal delivery was induced at 36 weeks gestation because of decreased fetal movements. The birth weight was 1.7 kg, birth length 45 cm, and head circumference 29 cm, all of which were less than their respective 10th percentiles. The physical examination of the neonate revealed brachycephaly, downslanted palpebral fissures, and maxillary hypoplasia. The anterior fontanel measured 1.5 × 2 cm; the cranial sutures were prominent. Cranial radiographs and cranial computed tomography demonstrated fusion of the coronal, sphenofrontal, and frontoethmoidal cranial sutures; the skeletal survey revealed osteopenia. A perimembranous ventricular septal defect (VSD) was also present. Further studies revealed (Figure 1B) hypercalcemia, hypophosphatemia, hyperalkaline phosphatasia, and an elevated level of parathyroid hormone (PTH). Serum concentrations of magnesium (1.0 mmol/L), calcitriol (369 pmol/L), and calcidiol (66.9 nmol/L) were normal for age. The spot urinary calcium/creatinine ratio was 0.8 mmol/mol creatinine. For management of hypercalcemia, the neonate
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Diaz-Thomas et al.: A novel CASR mutation associated with neonatal severe hyperparathyroidism 853
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Figure 1 (A) Pedigree of the family reported demonstrating autosomal recessive of NSHPT. (B) Table listing serum analyte concentrations in the reported family.
received furosemide, hydrocortisone, calcitonin, pamidronate, and a low calcium diet with temporary restoration of eucalcemia. At age 4 months because of unremitting hyperparathyroidism, patient 1 underwent subtotal parathyroidectomy. Immediately prior to surgery the physical examination revealed length 53.5 cm and weight 3.7 kg (less than their respective 3rd percentiles), brachycephaly, biparietal bossing, down-slanting palpebral fissures, and a Grade III/VI systolic murmur along the left sternal border. Preoperatively, the 24-h urinary calcium excretion was 0.68 mmol/mmol creatinine. After the surgical excision of 3.5 parathyroid glands, the iPTH levels declined and hypocalcemia developed requiring temporary supplementation with calcium gluconate (Figure 2). Serum calcium values
returned to the hypercalcemic range by the 13th postoperative day and persisted thereafter. The pathologic examination of the parathyroid glands revealed hyperplasia. The combined weight of three hyperplastic parathyroid glands was 160 mg (the normal weight of 4 parathyroid glands in a 3-month-old infant is approximately 25.4 mg). At age 23 months, patient 1 had delayed motor and speech development. He underwent the surgical repair of craniosynostosis at 49 months of age. At age 54 months, patient 1 remained hypercalcemic (2.92 mmol/L). Patient 2, the younger sister of patient 1, was the product of a 38-week uncomplicated pregnancy and vaginal delivery. The birth weight was 3.03 kg, length 50 cm, head circumference 34 cm. The physical examination at
3.375 Patient 1
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Figure 2 Changes in serum concentrations of calcium after subtotal parathyroidectomy in siblings with NSHPT.
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854 Diaz-Thomas et al.: A novel CASR mutation associated with neonatal severe hyperparathyroidism
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Figure 3 Site of the Gly768Val mutation of CASR in the family in which NSHPT was transmitted as an autosomal recessive characteristics.
birth disclosed brachycephaly, prominent cranial sutures, hypertelorism, and down-slanted palpebral fissures. At birth, patient 2 was hypercalcemic and hypophosphatemic and had high serum concentrations of PTH and 1,25-dihydroxyvitamin D (405 pmol/L, RR T point mutation at nucleotide c.2303 in exon 7 (c.2303G > T) resulting in the replacement of glycine by valine at codon 768 (p.Gly768Val = G768V) (Robert Thacker Ph.D., OCDEM, Genetics Laboratories, Headington, Oxford) (Figure 3). Concurrent sequencing of the CASR (GENE DX, Gaithersberg, MD, USA) confirmed the Gly768Val mutation and also revealed a homozygous variation at codon 986 (Ala986Ser), a common polymorphism that has been associated with alterations in circulating calcium levels in healthy adults (10). The homozygosity for this sequence variant has also been identified in a neonate with transient hypercalcemia (11). The genotyping of the CASR from peripheral monocytes of both parents, and
from a paraffin embedded parathyroid gland of patient 2 confirmed the Gly768Val mutation in these four subjects. Both children were homozygous, and both parents were heterozygous for the Gly768Val CASR variant.
Functional effect of mutant CASR The Gly768Val variant is situated in the extracellular loop of the CaSR that connects transmembrane domain 2 to transmembrane domain 3, an otherwise evolutionarily conserved site among mammals (Figure 3). The functional significance of the novel Gly768Val mutation was examined and compared to the wild type CASR and the CASR bearing an adjoining gain-of-function variant, Glu767Gln (6). When the Gly768Val, Glu767Gln, and the wild type CASRS were expressed in transfected HEK293 cells, protein was synthesized and migrated to the cell surface in each transfected cell line (Supplemental Figure 1). The functional significance of the Gly768Val CASR variant was further examined utilizing three computational programs (Polymorphism Phenotyping, version 2 (PolyPhen2) – http://genetics.bwh.harvard.edu/pph2; MutationTaster – http://mutationassessor.org; SIFT – http://sift-dna.org) that estimate the functional effect of a specific change in the amino acid sequence of a protein (7–9). PolyPhen2 evaluates both the amino acid sequence and the structure of the altered protein; PolyPhen2 gauged the Gly768Val variant of CASR to be “probably damaging” with a score of 1.0 (maximum). MutationTaster calculated a Functional Impact score of 3.345 for the Gly768Val variant of the CASR indicating that this mutation is likely to have an adverse effect on protein function of medium significance. The SIFT analysis predicted that the Gly768Val variant was “damaging.” The neighboring gain-of-function variant, Glu767Gln, was likewise assessed using the same three computational programs and also felt to produce an altered protein that would be deleterious to the function of the CaSR receptor.
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Diaz-Thomas et al.: A novel CASR mutation associated with neonatal severe hyperparathyroidism 855
Discussion Human CaSR is a 1078 amino acid (aa) protein containing three distinct regions: a large extracellular domain (aa 1-612), seven transmembrane domains (aa 613-862), and an intracellular tail domain (aa 863-1078) (5). The agonist binding in the cleft of the Venus flytrap (VFT) structure within the extracellular domain induces conformational changes leading to its closure and consequent three-dimensional changes in the transmembrane and tail domains of CaSR that activate intracellular signal transduction primarily through the Gαq and Gα11 subunits of G-protein (2, 12). The Gαq and Gα11 subunits stimulate phospholipase C activity, and the inositol phosphate signal transduction pathway leading to increased intracellular Ca2+ levels, and the activation of the mitogen-activating protein kinase signaling cascade eventuating in the regulation of gene transcription (13). The effects of inactivating mutations in the genes encoding a g-protein coupled receptor (GPCR) can be classified based on the functional consequence of the mutation: class I mutations interfere with receptor biosynthesis, class II mutations disrupt trafficking of the receptor to the cell surface, class III mutations interfere with ligand binding, class IV mutations impede receptor activation, and class V mutations have none of the above characteristics (14). In the majority of patients with familial hypocalciuric hypercalcemia (FHH), the disorder is transmitted as an autosomal dominant (monoallelic) trait likely explained by the dominant negative activity of the mutant CaSR receptor on the wild type CaSR (5). Inasmuch as the parents of the patients described in this report were asymptomatic and eucalcemic heterozygous carriers of the CASR mutation (Gly768Val) while the children bearing the mutation on both alleles (homozygous) were hypercalcemic with marked hyperparathyroidism, we conclude that NSHPT due to the Gly768Val variant of the CASR is transmitted as an autosomal recessive trait. Recently, the Gln459Arg inactivating mutation in the extracellular domain of CASR was found to be transmitted as an autosomal recessive disorder (15). The present study demonstrates that neither the Gly768Val loss-of-function mutation in the CASR nor the adjacent gain-of-function mutation (Glu767Gln) interferes with synthesis of the aberrant CaSR protein or its transit to the cell membrane in transfected human cells. The three computer programs that model the impact of an amino acid variant upon function of the parent protein predict that the Gly768Val mutation of CaSR will have an adverse effect. The biochemical and clinical effects of these mutations most likely lie in their interaction with
Ca2+, or in the manner in which they affect intracellular signaling. Further studies examining the spatial configurations of the mutant CaSRs and the effect of these alterations on dimerization of the variant receptors with the wild type CaSR, the binding affinities of the altered receptors for Ca2+, and their effects on the intracellular signal transduction pathways activated by the interaction of Ca2+ and the CaSR are required to identify the basic biochemical abnormalities these mutations confer upon the CaSR function. However, employing three computer programs that predict the effects of amino acid variations upon the configuration and function of the parent protein demonstrated that the CASR Gly768Val variant is likely to affect adversely the activity of the CaSR protein. Therapeutically, understanding the functional consequences of a mutation in the CaSR permits the identification of the patient who may respond to allosteric modifiers of the CaSR, i.e., a calcimimetic such as cinacalcet or a calcilytic agent. Indeed, a newborn with NSHPT associated with an Arg185Gln mutation in the CASR has been treated with cinacalcet with long-term normalization of the serum calcium level and suppression of serum PTH concentrations (16). Perhaps a calcimimetic agent would be useful in the management of the siblings described in this report. Another observation of interest is the presence of congenital craniosynostosis in the male subject, an abnormality that has not previously been recorded in patients with the CASR mutations. The CASR is expressed in the osteoblasts, osteocytes, and bone marrow stromal cells. The calcium acting through the CaSR inhibits the differentiation of osteoclasts and enhances the activity of osteoblasts (17). The CASR is also expressed in murine calvarial osteoblasts where it regulates their proliferative capacity acting through the Jun N-terminal kinase (JNK) pathway rather than the ERK signaling pathway (18, 19). Perhaps the inactivating mutation of CASR in the calvarial osteoblasts or osteocytes of one of our patients was of pathogenetic significance in the development of craniosynostosis. Further evaluation of the effect of the Gly768Val mutation of the CASR upon intracellular signal transduction pathways would be of interest. We conclude that NSHPT due to the Gly768Val variant of the CASR is transmitted as an autosomal recessive trait. This mutation does not interfere with the generation or movement of the protein product to the cell membrane. Acknowledgments: The authors thank Gary Litman, PhD, for enabling these studies to be conducted in his laboratory; Marci O’Driscoll and Larry Dishaw, PhD, for
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856 Diaz-Thomas et al.: A novel CASR mutation associated with neonatal severe hyperparathyroidism invaluable technical assistance; Dorothy Shulman, MD, E. Verena Jorgensen, MD, and Dewey Eichler, PhD, for helpful discussion.
Conflict of interest statement Authors’conflict of interest disclosure: The authors have no financial or competing interests to disclose. Research funding: The present work was conducted without any extramural funding.
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Supplemental Material: The online version of this article (DOI: 10.1515/jpem-2013-0343) offers supplementary material, available to authorized users.
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