Neonatal Hypocalcemic Seizures in Offspring of a Mother With Familial Hypocalciuric Hypercalcemia Type 1 (FHH1)

Abstract Context Familial hypocalciuric hypercalcemia type 1 (FHH1) is caused by loss-of-function mutations of the calcium-sensing receptor (CaSR) and is considered a benign condition associated with mild-to-moderate hypercalcemia. However, the children of parents with FHH1 can develop a variety of disorders of calcium homeostasis in infancy. Objective The objective of this work is to characterize the range of calcitropic phenotypes in the children of a mother with FHH1. Methods A 3-generation FHH kindred was assessed by clinical, biochemical, and mutational analysis following informed consent. Results The FHH kindred comprised a hypercalcemic man and his daughter who had hypercalcemia and hypocalciuria, and her 4 children, 2 of whom had asymptomatic hypercalcemia, 1 was normocalcemic, and 1 suffered from transient neonatal hypocalcemia and seizures. The hypocalcemic infant had a serum calcium of 1.57 mmol/L (6.28 mg/dL); normal, 2.0 to 2.8 mmol/L (8.0-11.2 mg/dL) and parathyroid hormone of 2.2 pmol/L; normal 1.0 to 9.3 pmol/L, and required treatment with intravenous calcium gluconate infusions. A novel heterozygous p.Ser448Pro CaSR variant was identified in the hypercalcemic individuals, but not the children with hypocalcemia or normocalcemia. Three-dimensional modeling predicted the p.Ser448Pro variant to disrupt a hydrogen bond interaction within the CaSR extracellular domain. The variant Pro448 CaSR, when expressed in HEK293 cells, significantly impaired CaSR-mediated intracellular calcium mobilization and mitogen-activated protein kinase responses following stimulation with extracellular calcium, thereby demonstrating it to represent a loss-of-function mutation. Conclusions Thus, children of a mother with FHH1 can develop hypercalcemia or transient neonatal hypocalcemia, depending on the underlying inherited CaSR mutation, and require investigations for serum calcium and CaSR mutations in early childhood.

F amilial hypocalciuric hypercalcemia type 1 (FHH1) is an autosomal dominant disorder caused by lossof-function mutations of the calcium-sensing receptor (CaSR), which is encoded by the CASR gene on chromosome 3q21.1 (1). The CaSR regulates calcium homeostasis by enhancing intracellular calcium (Ca 2+ i ) mobilization and stimulating the mitogen-activated protein kinase (MAPK) cascade (2), which in turn reduces parathyroid hormone (PTH) secretion and increases urinary calcium excretion. FHH1 is generally benign and characterized by mild-to-moderate elevations of serum calcium, normal or mildly raised serum PTH, and a calcium-to-creatinine clearance ratio of less than 0.01 (1). However, the offspring of individuals with FHH1 may manifest a variety of disturbances of calcium homeostasis (3). Thus, children inheriting biallelic loss-of-function CaSR mutations from FHH1 parents can develop neonatal severe primary hyperparathyroidism (4). In addition, children inheriting a monoallelic loss-of-function CaSR mutation from the father are at risk of neonatal severe primary hyperparathyroidism if the mother is normocalcemic, whereas they would likely develop the more benign phenotype of FHH1 if they inherited the loss-of-function CaSR mutation from the mother (5), and can occasionally be normocalcemic because of incomplete penetrance of the CaSR mutation (6). Furthermore, maternal hypercalcemia caused by FHH1 may potentially cause transient hypoparathyroidism in the unaffected offspring (3), and such hypocalcemia has been reported in 2 unrelated neonates born to mothers with clinically diagnosed FHH (7,8), although the CASR mutation status in these cases was not established. Here, we report a 3-generation FHH1 kindred with a novel loss-of-function CASR mutation (p.Ser448Pro), in which an affected mother, who was heterozygous for the CaSR mutation, had 4 children, 2 of whom had asymptomatic hypercalcemia in association with the heterozygous CaSR Ser448Pro mutation, 1 without a CaSR mutation was normocalcemic, whereas another, also without a CaSR mutation, had hypocalcemic seizures as a consequence of transient neonatal hypoparathyroidism.

Participants
The three generation FHH1 kindred comprised a man (individual I.1, Fig. 1A Investigations showed a low serum adjusted-calcium of 1.57 mmol/L (6.28 mg/dL), elevated serum phosphate of 3.45 mmol/L (10.7 mg/dL); normal, 1.30 to 2.60 mmol/L (4.0-8.0 mg/dL), and an inappropriately normal serum PTH of 2.2 pmol/L; normal, 1.0 to 9.3 pmol/L ( Fig. 1A and 1B). Serum magnesium was borderline low at 0.62 mmol/L (1.5 mg/dL); normal, 0.62 to 1.00 mmol/L (1.5-2.4 mg/dL), and serum 25-hydroxyvitamin D was low at 27 nmol/L; adequate is greater than 50 nmol/L. These findings indicated that she had hypoparathyroidism and vitamin D deficiency. She did not have clinical or biochemical features of the Di George or hypoparathyroidism-deafness-renal anomalies syndromes (9). She was treated with intravenous infusions of 10% calcium gluconate (0.5 mL/kg over 30 minutes), magnesium sulfate (100 mg/kg over 10 minutes), phenobarbitone (5 mg/kg on 3 consecutive days), and also given cholecalciferol (1000 IU daily). Over the next 48 hours her serum calcium concentration increased into the normal range (Fig. 1B). This rapid improvement in serum calcium suggested a transient cause for the hypoparathyroidism, most likely secondary to the maternal hypercalcemia. She was discharged at age 3 weeks with normal serum calcium (Fig. 1B) and no further symptoms.

Mutational analysis
Analysis of the CASR gene was performed using leukocyte DNA following informed consent. Sanger sequencing of all coding exons and exon-intron boundaries was undertaken with exon-specific primers (Sigma Aldrich), and using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Life Technologies), and an automated detection system (ABI3730 Automated capillary sequencer; Applied Biosystems), as reported (10,11). Investigation of potentially pathogenic variants was undertaken using the publicly accessible Genome Aggregation Database (gnomAD) database: (https:// gnomad.broadinstitute.org/), which is a dataset comprising 125 748 exome sequences and 15 708 wholegenome sequences from unrelated individuals.

Western blot analyses
Expression of WT and mutant proteins by the pEGFP-N1-CaSR constructs was assessed by Western blot analysis, with the calnexin housekeeping protein being used as a loading control. For blots under reducing conditions, lysates were mixed with Laemmli loading buffer containing β-mercaptoethanol (BioRad) and run in Tris-Glycine-sodium dodecyl sulfate buffer. For blots under nonreducing (native) conditions, lysates were mixed with Laemmli buffer without β-mercaptoethanol and run in Tris-Glycine buffer. The plasma membrane protein fraction was isolated from cells transfected with pEGFP-N1-CaSR constructs using a plasma membrane extraction kit (Abcam, catalog No. 65400), as described (16). Plasma membrane calcium ATPase (PMCA1) protein was used as a loading control for plasma membrane fractions (16). The following primary antibodies were used for Western blot analysis: anti-CaSR (ADD, ab19347, Abcam), anticalnexin (MA3-027, Thermo Fisher Scientific) and anti-PMCA1 (ab190355, Abcam). The Western blots were visualized using an Immuno-Star WesternC kit (BioRad) on a BioRad Chemidoc XRS+ system (11,14,15). Densitometry was performed using ImageJ (National Institutes of Health) and analyzed using GraphPad Prism. CaSR protein abundance was normalized to the protein loading control and expressed relative to WT CaSR. Statistical analysis was performed using the student t test.

Functional assays
Luciferase reporter assays were undertaken to measure CaSR-mediated SRE and NFAT responses, as previously described (14,15). Cells were transiently transfected with 100 ng/ml of the WT or mutant pEGFP-N1-CaSR constructs, 100 ng/ml luciferase construct (either pGL4-NFAT or pGL4-SRE) and 10 ng/ml pRL null control luciferase reporter. At 48 hours post-transfection, cells were then treated with 0.1 to 10 mM CaCl 2 and incubated for 4 hours before lysis and measurement of luciferase activity using Dual-Glo Luciferase (Promega) on a Veritas Luminometer (Promega), as previously described (14). Luciferase to renilla ratios were expressed as fold-changes relative to responses at basal CaCl 2 concentrations (0.1 mM). All assay conditions were performed in 4 to 8 biological replicates. Nonlinear regression of the concentrationresponse curves was performed with GraphPad Prism (GraphPad) to calculate the half-maximal (EC 50 ) and maximal (E max ) responses for each separate experiment. Statistical analysis was performed by 2-way ANOVA with Tukey multiple-comparisons test, and significant alterations in EC 50 values were assessed using the F test (17).

Results
DNA sequence analysis identified a novel germline heterozygous CASR variant (c.1342 T > C; p.Ser448Pro) in the hypercalcemic children, mother, and grandfather (Fig. 1C). However, this CASR variant was absent in the hypocalcemic infant and her normocalcemic sibling. The p.Ser448Pro variant was shown to affect an evolutionarily conserved residue (Fig. 1D), and this variant was not detected in the gnomAD database. The WT Ser448 residue is located in the CaSR ECD (Fig. 2). The structural consequences of the p.Ser448Pro variant were assessed using a reported crystal structure of the dimeric CaSR ECD (Fig. 2) (13). The introduction of the variant Pro448 CaSR residue was predicted to disrupt a hydrogen bond interaction across the CaSR dimer interface involving the CaSR ECD Thr445 and Lys52 residues, and also potentially affect a disulfide bond formed between the neighboring Cys449 and Cys437 residues (Fig. 2). To determine the effects of these predicted structural changes on CaSR-mediated signaling, HEK293 cells were transiently transfected with pEGFP-N1-CaSR constructs expressing WT (Ser448) or variant (Pro448) CaSR proteins. A reported FHH1-causing (Leu173Pro) CaSR protein (Fig. 3) was used as a control loss-of-function mutation (17). CaSR expression was confirmed by fluorescence microscopy and Western blot analysis (Fig. 3A and 3B). Western blot analysis was also performed under nonreducing conditions to detect dimeric CaSR proteins, and on plasma membrane protein fractions of cells transiently transfected with pEGFP-N1-CaSR constructs ( Fig. 3C and 3D). Significant alterations were not detected in the expression of total cellular, dimeric, or plasma membrane forms of the Pro448 and Pro173 CaSR proteins compared to WT CaSR (Fig. 3E).
The signaling responses of these CaSR-expressing cells were assessed following stimulation with extracellular calcium (Ca 2+ e ) using luciferase reporter assays for NFAT and SRE, which are downstream indicators of Ca 2+ i mobilization and MAPK signaling, respectively (17). The NFAT and SRE responses of the CaSRexpressing cells increased in a concentration-dependent manner following stimulation with increasing Ca 2+ e concentrations ( Fig. 4A and 4B). However, cells expressing the variant (Pro448) CaSR protein or the FHH1-causing (Pro173) CaSR protein showed significantly reduced NFAT and SRE responses compared to WT cells (Fig. 4A and 4B). Indeed, cells expressing the Pro448 CaSR protein showed a rightward shift in the NFAT and SRE concentration-response curves with significant increases in the respective EC 50 values when compared to WT CaSR ( Fig. 4A and 4B), whereas cells expressing the Pro173 CaSR protein showed significant reductions in the maximal NFAT and SRE foldchange responses compared to WT CaSR ( Fig. 4A and  4B). Thus, functional studies of the p.Ser448Pro CaSR variant demonstrate this to represent a loss-of-function CaSR mutation.

Discussion
These findings, which have identified a novel p.Ser448Pro CaSR mutation in an FHH1 kindred, demonstrate that the infant of a mother with FHH1 can be hypercalcemic, normocalcemic, or hypocalcemic, depending on the inheritance of the maternal CaSR mutation. Thus, children harboring a maternally inherited loss-of-function CaSR mutation typically have an FHH1 phenotype with asymptomatic hypercalcemia during infancy, whereas children who have not inherited a loss-of-function CaSR mutation from a mother with FHH1 may either be normocalcemic or develop transient neonatal hypocalcemia, which can be symptomatic and cause seizures. However, not all unaffected infants Figure 2. Ribbon diagram of the dimeric calcium-sensing receptor (CaSR) extracellular domain (ECD), which is derived from a published crystal structure (13). The CaSR ECD comprises a bilobed venus-fly-trap domain and a cysteine-rich domain (CRD). The WT Ser448 residue (purple) is located in lobe 1 of the CaSR ECD. The Leu173 residue, which is the site of a reported familial hypocalciuric hypercalcemia type 1 (FHH1)-causing Leu173Pro mutation (17), is shown in yellow. A close-up view shows the wild-type (WT) Ser448 residue (purple) to be in close proximity to a Thr445 residue, which is located at the extracellular dimer interface. The Thr445 residue forms a hydrogen bond (red dashed line) with the Lys52 residue of the neighboring CaSR protomer. The introduction of a mutant Pro448 residue (red) is predicted to sterically interfere with the Thr445 residue, thereby disrupting the Thr445-Lys52 interaction. The WT Ser448 residue is also located near a Cys449-Cys437 disulfide bond (yellow), and the p.Ser448Pro mutation may disrupt this interaction.
Downloaded from https://academic.oup.com/jcem/article-abstract/105/5/dgaa111/5801090 by guest on 16 April 2020 of FHH1 mothers will develop symptomatic hypocalcemia, as highlighted by the unaffected normocalcemic son (individual III.1, Fig. 1A), who remained well during infancy. This suggests that additional factors, such as vitamin D deficiency, could have contributed to the marked hypocalcemia in the daughter (individual III.4, Fig. 1A). Transient hypocalcemia affects around 1 in 50 term neonates (18), and can cause irritability and seizures (19). It is commonly associated with vitamin D deficiency and low or inappropriately normal PTH concentrations (18), both of which were features of this case. Reduced PTH secretion in the neonatal period can arise from delayed parathyroid gland maturation, hypomagnesemia (18), or as a consequence of maternal hypercalcemia, which suppresses fetal parathyroid activity (19). The major cause of maternal hypercalcemia is primary hyperparathyroidism, which affects approximately 1 out of 3000 women of reproductive age (20). In contrast, FHH1 has rarely been reported as a cause of hypercalcemia in pregnancy, and this condition may remain asymptomatic throughout gestation (21). However, it is possible that some mothers with FHH1 may develop more severe hypercalcemia in the third trimester because of increases in circulating PTH-related peptide concentrations (22), and therefore warrant serum calcium monitoring during the later stages of pregnancy. In addition, the present case indicates that maternal FHH1 poses a risk to the unaffected neonate because of suppression of the parathyroid gland function in utero. The risk of transient neonatal hypoparathyroidism is likely related to the degree of maternal hypercalcemia, and it is notable that the maternal serum calcium concentration in this case, and the 2 reported cases of FHH-associated neonatal hypocalcemia (7,8), was substantially elevated at greater than 2.90 mmol/L (> 11.6 mg/dL). These findings indicate the importance of assessing maternal serum calcium as part of the evaluation of an infant with hypocalcemia, particularly because this may unmask the presence of FHH in the mother. Furthermore, serum calcium should be assessed at birth in the infant of a mother with FHH1 and also during the first 1 to 2 weeks after birth, and CaSR mutational analysis considered for the newborn offspring of FHH1 parents. The p.Ser448Pro mutation identified in this FHH1 family is located within the CaSR ECD. This region of the CaSR binds a range of extracellular ligands, which include calcium, magnesium, and amino acids (13). However, our structural analysis showed that the mutated Ser448 residue is not located at one of the ECD ligand binding sites, but instead contributes to the ECD dimer interface, which is involved in agonist-mediated activation of the CaSR, and reported to be a hotspot for loss-of-function CaSR mutations (13,17). Although the p.Ser448Pro mutation did not significantly alter the abundance of dimeric CaSR protein, our functional studies demonstrated the p.Ser448Pro mutation to cause a loss of function by impairing CaSR-mediated signaling via the Ca 2+ i and MAPK pathways. The p.Ser448Pro mutation was associated with a greater than 30% increase in the EC 50 value when compared to WT both in the in vitro NFAT and SRE assays ( Fig. 4A and AB), and this degree of loss of function is similar to that reported for other FHH1-causing CaSR mutations (17).
In summary, our studies have a identified a novel loss-of-function CaSR mutation that caused asymptomatic hypercalcemia in a mother and her children who had inherited the mutation, but was also associated with transient neonatal hypocalcemic seizures in one of her children who had not inherited the CaSR mutation. ] e responses and shown as mean ± SEM of 4 to 8 biological replicates. The half-maximal (EC 50 ) and maximal fold-change values are shown below the concentration-response curves. NS, nonsignificant, ****P less than .0001, ***P less than .001, **P less than .01, *P less than .05 compared to WT.