HYPERCALCEMIA (FHH) IN A YOUNG HISPANIC MAN. Running title: New mutation Calcium Sensing Receptor causing FHH.

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1 AACE Clinical Case Reports Rapid Electronic Articles in Press Rapid Electronic Articles in Press are preprinted manuscripts that have been reviewed and accepted for publication, but have yet to be edited, typeset and finalized. This version of the manuscript will be replaced with the final, published version after it has been published in the print edition of the journal. The final, published version may differ from this proof. Case Report ACCR A NOVEL MUTATION IN CALCIUM SENSING RECEPTOR PRESENTING AS FAMILIAL HYPOCALCIURIC HYPERCALCEMIA (FHH) IN A YOUNG HISPANIC MAN. Parvathy Madhavan, MD 1, Thi Hong Van Do, MD 1, Allen Bale MD 3, Sachin Majumdar, MD 1,2 From: 1 Departments of Internal Medicine and 2 Endocrinology, Bridgeport Hospital, Yale New Haven Health System, CT USA. 3 Yale University School of Medicine, New Haven, CT, USA. Running title: New mutation Calcium Sensing Receptor causing FHH. Corresponding author: Parvathy Madhavan, MD Section of Endocrinology Department of Internal Medicine Bridgeport Hospital, Yale New Haven Health System 267 Grant Street, Bridgeport, CT madhavan.parvathy@gmail.com

2 Abstract Objective: Familial hypocalciuric hypercalcemia (FHH) is considered a relatively benign condition characterized by elevated serum calcium with relatively low urinary calcium excretion. It typically results from an altered set point in calcium homeostasis originating from mutations in the Calcium Sensing Receptor (CASR), AP2S1, or GNA11 genes, which encode for the calcium sensing receptor (CASR), adaptor-related protein complex 2, and G-protein alpha-11 subunit, respectively. Despite numerous reports of novel variants in these genes associated with FHH new variants continue to be discovered. Methods: We describe a 20 year old man with a family history of hypercalcemia who had clinical findings compatible with FHH, and no evidence of multiple endocrine neoplasia (MEN), who underwent CASR gene sequencing for evaluation of hypercalcemia. Parathyroid SPECT (Single Proton Emission Computerized Tomography ) scan was normal. Results: CASR gene sequencing revealed a previously unreported heterozygous intronic variant at position A>G (chr 3: ) resulting in a 77 residue deletion. His mother by report has a history of bipolar disorder and hyperparathyroidism with an adenoma found on imaging, yet our patient had no evidence of adenoma and therefore no surgical intervention was recommended. Given that the CASR plays a role in parathyroid growth one might consider that some variants in the CASR may ultimately lead to parathyroid hypertrophy and be mistaken for primary hyperparathyroidism.

3 Conclusion: Longer term clinical follow up will be helpful in understanding the ultimate effects of specific mutations on parathyroid growth or progression to significant hypercalcemia. Abbreviations: FHH = Familial hypocalciuric hipercalcemia; CASR = Calcium Sensing Receptor; MEN = Multiple endocrine neoplasia; dbscsnv = Database for Single Nucleotide Variants within splicing consensus regions; SPECT = Single Proton Emission Computerized Tomography; PTH = Parathyroid hormone; TNFα = Tumor necrosis factor alpha; IL1-6 = Interleukin.

4 Introduction The calcium sensing receptor is a 1,078 amino acid G protein coupled receptor that has 3 domains - extracellular, transmembrane and intracellular, and the gene localizes to chromosome 3q and has 8 exons, the first (1A and 1B) encoding an alternative 5 - untranslated splicing region [1-4]. The promoter of CASR gene is responsive to 1,25-OH vitamin D, TNFα and IL1-6 [5]. The CASR plays a key role in regulating calcium homeostasis by sensing minute changes in serum calcium concentration and regulating PTH (Parathyroid hormone) secretion as well as renal tubular calcium reabsorption [6, 7]. It is present in the chief cells of parathyroid glands as well as bone, kidney, and gut, among other tissues [8]. Mutations of the CASR can lead to loss or gain of function resulting in hypercalcemia and hypocalcemia, respectively [1, 7, 8]. Inactivating functional mutations of the CASR result in autosomal dominant familial benign hypercalcemia, or familial hypocalciuric hypercalcemia (FHH) syndrome type 1 (mostly heterozygous mutations) and neonatal severe hyperparathyroidism (homozygous mutations), whereas gain of function mutations can cause a form of autosomal dominant hypoparathyroidism and sporadic hypoparathyroidism [1, 7, 9]. While numerous variants in the CASR, and other genes associated with FHH have been identified, we report a novel mutation in the CASR gene associated with the clinical and biochemical phenotype of FHH. Case history A 20-year-old Hispanic man was referred for evaluation of hypercalcemia and hypertension. He denied having fatigue, polyuria, polydipsia, abdominal symptoms or

5 confusion, and had no prior history of jaw tumors or kidney stones. His mother is currently being evaluated for parathyroidectomy at another institution due to hypercalcemia and neurocognitive symptoms in association with a parathyroid adenoma on imaging. No other family members have a known history of hypercalcemia, kidney stones, or renal dysfunction. On exam he was afebrile, BP 124/80 mm hg, HR 70 bpm, BMI 23.96kg/m2. Physical exam was within normal limits and laboratory testing was undertaken to evaluate for evidence of MEN syndromes given his age and reported history of hypertension. Laboratory findings Biochemistry is shown in tables 1-3. His parathyroid hormone was inappropriately normal in the setting of hypercalcemia but there were no hormonal findings to suggest an MEN syndrome (Table 3). He had a family history of hypercalcemia and his estimated calcium to creatinine clearance ratio was below 0.01 supporting familial hypocalciuric hypercalcemia (FHH) rather than primary hyperparathyroidism [1]. Further a Parathyroid SPECT scan did not show evidence of parathyroid adenoma. However, since there can be biochemical overlap, and given that his mother carried a diagnosis of primary hyperparathyroidism and reportedly had a parathyroid adenoma, we pursued genetic testing. This revealed a heterozygous intronic variant in the CASR gene, NM_00038:c A>G (chr 3: ) [hg19], which occurs in the splicing site between intron 5 for the extracellular domain. Discussion Many loss of function mutations in the CASR, as seen in FHH, cluster around aspartate and glutamate rich regions (codons 39 to 300), within the extracellular domain [4]. Genetic

6 testing of CASR gene in our patient revealed a previously unreported heterozygous intronic variant at position A>G (chr 3: ) resulting in a 77-residue deletion. This variant is predicted to reduce the splicing efficiency by dbscsnv (Database for Single Nucleotide Variants within splicing consensus regions) [10]. Only 2% of the CASR gene mutations are in the splicing area so it is considered to be very rare mutation [11]. A different variant at the same location (c a>c) has been previously reported in a family with FHH type 1 [11]. The biochemical findings in our patient in addition to the change at position A>G are suggestive of FHH type 1. Approximately 65% of FHH cases are transmitted in an autosomal dominant manner resulting in heterozygous mutations of the CASR and are characterized by moderate asymptomatic hypercalcemia as in our patient. However, hypercalcemia can be marked in 10 % of cases [1, 4]. As seen in our case PTH is inappropriately normal in 80% of cases in relation to the hypercalcemia and some patients also demonstrate moderate hypermagnesemia and a positive family history [1]. Additionally, increased prevalence of chondrocalcinosis and occasional cases of acute pancreatitis and gallstones have been reported in association with FHH [4]. These findings and events have not been observed in our patient but longer-term follow-up will be of interest, especially given the possibility that some inactivating mutations in the CASR can result in hyperplasia [12] and his mother is reported to have an adenoma. There is evidence that heterozygous inactivation mutations of CASR are associated with parathyroid gland hyperplasia in a mouse model [13]. A study in bovine cells showed that the CASR co-localizes with caveolin-1, and that changes in caveolin expression are

7 associated with formation of parathyroid neoplasms [14]. The CASR is a not only involved in calcium homeostasis, PTH secretion, and Vitamin D synthesis, but it is also has non calciotropic functions and is expressed in other tissues and systems like the cardiovascular, central nervous, and respiratory systems [15]. The physiologic function of the CASR is currently being the investigated for its potential role in pathology of hypertension, vascular calcification, asthma, Alzheimer s disease and bone metastasis due to this extensive expression [15]. The CASR may also play a paradoxical role as tumor suppressor gene in some cancers like neuroblastoma, colon and parathyroid cancer, and as an oncogene in malignancies like breast and prostate cancer and long term follow up of the patient with this CASR mutation will provide us valuable information [15]. Hence, when individuals with FHH are identified genetic testing can be helpful in relating clinical phenotypes to specific variants that may impact other aspects of health, or be associated with differing clinical courses such as the development of adenomas. Our patient had a mutation located in a splice region predicted to lead to an in-frame deletion within the extracellular domain of the CASR, yet more than 85% of CASR mutations are missense substitutions while deletion, non-sense, insertion, and splice-site variants are rare [3, 16, 17]. There have been several case reports describing patients with FHH who were also noted to have parathyroid adenoma [18-20]. Typically surgical management of the adenoma is not recommended, but Burski et al describes a 45 year old woman who had FHH and a large, seemingly functional parathyroid adenoma which was surgically

8 removed and the serum calcium and PTH values were noted to fall abruptly and stabilize at a level characteristic of FHH [18]. Our patient s mother, who we were unable to test, had been diagnosed with an adenoma yet she may have the same mutation and it may be associated with adenoma development. Therefore, it is important to report and follow the clinical course of patients with variants in the CASR gene as they can enrich our knowledge of what to expect in the natural history of FHH, contribute to clinical decision making, and perhaps provide insight into the role of the CASR beyond calcium homeostasis alone. References 1. Vahe C, Benomar K, Espiard S, et al. Diseases associated with calciumsensing receptor. Orphanet J Rare Dis. 2017;12: Hannan FM, Nesbit MA, Christie PT, et al. A homozygous inactivating calciumsensing receptor mutation, Pro339Thr, is associated with isolated primary hyperparathyroidism: correlation between location of mutations and severity of hypercalcaemia. Clin endocrinol. 2010;73: Hannan FM, Nesbit MA, Zhang C, et al. Identification of 70 calcium-sensing receptor mutations in hyper-and hypo-calcaemic patients: evidence for clustering of extracellular domain mutations at calcium-binding sites. Hum Mol Genet. 2012;21: Thakker R. Calcium-sensing receptor: Role in health and disease. Indian J Endocrinol Metab. 2012;16:S213.

9 5. Canaff L, Hendy GN. Calcium-sensing receptor gene transcription is upregulated by the proinflammatory cytokine, interleukin-1β role of the NF-κB pathway and κb elements. J Biol Chem. 2005;280: Brown EM. Clinical lessons from the calcium-sensing receptor. Nat Rev Endocrinol. 2007;3: Vargas-Poussou R, Huang C, Hulin P, et al. Functional characterization of a calcium-sensing receptor mutation in severe autosomal dominant hypocalcemia with a Bartter-like syndrome. J Am Soc Nephrol. 2002;13: Tfelt-Hansen J, Brown EM. The calcium-sensing receptor in normal physiology and pathophysiology: a review. Crit Rev Clin Lab Sci. 2005;42: Yamauchi M, Sugimoto T, Yamaguchi T, et al. Familial Hypocalciuric Hypercalcemia Caused by an R648stop Mutation in the Calcium Sensing Receptor Gene. J Bone Miner Res. 2002;17: Liu X, Wu C, Li C, Boerwinkle E. dbnsfp v3. 0: A one stop database of functional predictions and annotations for human nonsynonymous and splice site SNVs. Hum Mutat. 2016;37: Vargas-Poussou R, Mansour-Hendili L, Baron S, et al. Familial hypocalciuric hypercalcemia types 1 and 3 and primary hyperparathyroidism: similarities and differences. J Clin Endocrinol Metab. 2016;101: Fukumoto S, Chikatsu N, Okazaki R, et al. Inactivating mutations of calciumsensing receptor results in parathyroid lipohyperplasia. Diagn Mol Pathol. 2001;10:242-7.

10 13. Ho C, Conner DA, Pollak MR, et al. A mouse model of human familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Nat Genet. 1995;11: Kifor O, Kifor I, Moore Jr FD, et al. Decreased expression of caveolin-1 and altered regulation of mitogen-activated protein kinase in cultured bovine parathyroid cells and human parathyroid adenomas. J Clin Endocrinol Metab. 2003;88: Diaz-Soto G, Rocher A, García-Rodríguez C, Núñez L, Villalobos C. The calcium-sensing receptor in health and disease. Int Rev Cell Mol Biol. 327: Elsevier; Hannan FM, Thakker RV. Calcium-sensing receptor (CaSR) mutations and disorders of calcium, electrolyte and water metabolism. Best Pract Res Clin Endocrinol Metab. 2013;27: Hendy GN, Canaff L, Cole DE. The CASR gene: alternative splicing and transcriptional control, and calcium-sensing receptor (CaSR) protein: structure and ligand binding sites. Best Pract Res Clin Endocrinol Metab. 2013;27: Burski K, Torjussen B, Paulsen A, Boman H, Bollerslev J. Parathyroid adenoma in a subject with familial hypocalciuric hypercalcemia: coincidence or causality? J Clin Endocrinol Metab. 2002;87: Forde HE, Hill AD, Smith D. Parathyroid adenoma in a patient with familial hypocalciuric hypercalcaemia. BMJ case reports. 2014;2014:bcr Brachet C, Boros E, Tenoutasse S, et al. Association of parathyroid adenoma and familial hypocalciuric hypercalcaemia in a teenager. Eur J Endocrinol 2009;161:

11 Table 1. Biochemistry Blood Ref. Range Nov-16 Oct-17 Sodium mmol/l 140 Potassiu (H) m mmol/l 4.4 Chloride mmol/l 100 CO mmol/l 27 GFR >60mL/m in/1.73 m2 > Creatinine 1.25 mg/dl 1.27 (H) 1.21 Glucose mg/dl 77 Calcium (H) mg/dl 10.9 (H) Calcium, (H) Ionized mg/dl Magnesiu m mg/dl Phosphor us mg/dl H= High or above normal range, L= Low or below normal range

12 Table hours urine 24 hours Ref. urine Range Dec-17 Volume, Timed Urine ml 2025 Creatinine , Total in g/24hrs Sample 2.04 Creatinine, Timed mg/dl Urine Calcium, Timed mg/d 6.00 (L) Urine L Calcium, 100- Total in 300mg/T Sample V H= High or above normal range, L = low or below normal range. Fractional excretion of calcium =

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