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.

Keywords: calcium-sensing receptor; hypercalcemia; hypocalcemia; hypoparathyroidism; loss-of-function; mutation.

Figure 1.

A, Pedigree and biochemistry of the family with familial hypocalciuric hypercalcemia type 1 (FHH1). Male and female family members are represented by squares and circles, respectively. Affected and unaffected individuals are represented by filled and open symbols, respectively. The hypocalcemic infant is shown in gray. B, Serum concentrations of adjusted-calcium, phosphate, and parathyroid hormone (PTH) in the hypocalcemic infant during the first 70 days postpartum. The red box indicates period of treatment with intravenous calcium and magnesium. Normal ranges are indicated by the gray areas. C, A heterozygous T > C transition at nucleotide c.1342 was identified in the hypercalcemic family members, which changes a TCC codon to CCC and is predicted to result in a missense amino acid substitution from Ser to Pro at position 448 in the CaSR protein. D, Multiple protein sequence alignment showing evolutionarily conservation of the calcium-sensing receptor (CaSR) Ser448 residue (bold). The gray area indicates conserved CaSR residues. The mutant (m) Pro448 residue is shown in red. 25OHD, 25-hydroxyvitamin D; CCCR, calcium-to-creatinine clearance ratio; sCa, serum calcium.

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.

Figure 3.

A, Fluorescence microscopy of HEK293 cells transiently transfected with pEGFP-N1–calcium-sensing receptor (CaSR) constructs expressing wild-type (WT) (Ser448) or mutant (m) (Pro448) CaSR proteins, or a known familial hypocalciuric hypercalcemia type 1 (FHH1)-causing (Leu173Pro) mutant CaSR protein. Green fluorescent protein expression in these cells indicates successful transfection and expression by these constructs. Bar indicates 10 μm. B to D, Western blot analysis of cells transiently transfected with WT or mutant CaSR proteins. B, Analysis of whole-cell lysates under reducing conditions. Calnexin was used as a loading control and the blot shown is representative of n = 5 independent experiments. C, Analysis of whole-cell lysates under nonreducing conditions to detect dimeric CaSR. Calnexin was used as a loading control and the blot shown is representative of n = 3 independent experiments. D, Western blot analysis of plasma membrane fractions. Plasma membrane calcium ATPase (PMCA1) was used as a loading control and the blot shown is representative of n = 6 independent experiments. E, Densitometric analysis of relative CaSR abundance shown as mean ± SEM. NS, nonsignificant.

Figure 4.

Concentration-response curves of extracellular calcium (Ca²⁺_e)-induced: A, nuclear factor of activated T-cells (NFAT) and B, serum-response element (SRE) luciferase reporter responses of HEK293 cells expressing wild-type (WT) (black) or mutant (Pro448, red) calcium-sensing receptor (CaSR) proteins, or a known familial hypocalciuric hypercalcemia type 1 (FHH1)-causing (Leu173Pro, gray) CaSR mutant protein. Responses at each Ca²⁺_e concentration are expressed as a fold-change of basal [Ca²⁺]_e responses and shown as mean ± SEM of 4 to 8 biological replicates. The half-maximal (EC₅₀) 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.