PTH infusion ameliorates seizures in autosomal dominant hypocalcemia type 1

a of pediatric normal ranges; his help with assessments of nephrocalcinosis.

). She required multiple i.v. boluses of calcium and magnesium which provided temporary amelioration of the seizures, and she was treated with phenobarbitone for 2 weeks, which was discontinued after her serum calcium had normalised on CSPI, following which she remains free of seizures.
Before CSPI: She was treated with high dose oral alfacalcidol, cholecalciferol, calcium and magnesium supplementation, and i.v. boluses and infusion of calcium (Fig. 1A). However, her seizures persisted.
After CSPI: She started CSPI at 5 weeks of age, which normalised the serum calcium and abolished the seizures. She required short admissions for asymptomatic hypocalcemia due to cannula failure or intercurrent illnesses. Her serum creatinine has remained stable on CSPI and there is no evidence of nephrocalcinosis. She was walking and talking at an appropriate age, and is growing normally. Bone mineral apparent density (BMAD) at the age of 3 yrs is within the age-adjusted pediatric reference interval (Fig. S4).

Patient 2
Presentation: Patient 2 was born at 35 weeks gestation with a birth weight of 3.01 kg, and presented with bronchiolitis and seizures at 28 days of age. He had hypocalcemia, hypomagnesemia, hyperphosphatemia, and low circulating PTH concentrations (Fig. 1A). His parents are of White European origin. There was no family history of hypocalcemia.
Before CSPI: He was treated with oral alfacalcidol, and calcium and magnesium supplementation (Fig. 1A), and was admitted to hospital 1-2 times per week with symptoms of hypocalcemia (tetany). He also had mild polyuria and polydipsia, consistent with hypercalciuria, and by the age of 10 months had developed bilateral severe nephrocalcinosis.
After CSPI: He started CSPI treatment at 10 months of age. He had one more seizure on CSPI and 3 hospital admissions related to hypocalcemia with mild tetany in feet or hands. His creatinine concentrations have been stable, urinary calcium excretion normalised after first year of therapy, and nephrocalcinosis has not progressed. His development is normal, and he is growing normally. BMAD at the age of 3 yrs is within the age-adjusted pediatric reference interval (Fig. S4).

Patient 3
Presentation: Patient 3 was born to a mother with ADH1. Antenatal scans showed polyhydramnios. During pregnancy, her mother required temporary dialysis and developed seizures. She was delivered at the 31 st gestational week by cesarean section for maternal reasons with a birth weight of 1.14 kg (3 rd centile). Parents are of White European origin. She presented with respiratory depression at birth that required mechanical ventilation and was found to have severe hypocalcemia (serum calcium 0.76 mmol/L) and hypomagnesemia (0.52 mmol/L). Despite i.v. calcium boluses and infusion, she developed hypocalcemic seizures at the age of 5 days.
Before CSPI: She was treated with alfacalcidol, and calcium and magnesium supplementation ( Fig. 1A), but still experienced multiple seizures and required frequent hospital admissions.
She developed nephrocalcinosis and renal impairment with increased creatinine concentrations from 2 years of age and her GFR was 50-60 ml/1.73m 2 /min in the years before start of CSPI.
At the age of 18 years she commenced PTH(1-84) bolus therapy, but suffered hypocalcemic seizures whilst on once daily PTH(1-84) injections. She was switched to twice daily PTH(1-34) bolus injections in an attempt to improve this, and showed some reduction in the frequency of seizures and reduced hospitalizations. She had mild learning difficulties requiring special education. She completed normal growth.  1A). There was no family history of hypocalcemia.
Before CSPI: He had further recurrent left-sided focal seizures aged 5 weeks despite being on alfacalcidol, and calcium and magnesium supplementation (Fig. 1A), and received further treatment with phenobarbitone. He developed bilateral cataracts by 6 weeks of age, which were treated with lensectomy and glasses.
After CSPI: CSPI was started at age 7 weeks and his seizures completely stopped, he required 3 further hospital admissions due to intercurrent illness and infection of the infusion site. He has not developed nephrocalcinosis and his renal function is normal. His development, learning, and growth are normal. BMAD at age of 3 yrs is within the age-adjusted pediatric reference interval (Fig. S4).

Patient 5
Presentation: Patient 5 was born at 38 weeks gestation after induction of labour and emergency caesarean section due to polyhydramnios, with a birth weight of 4.15 kg. Parents are of Black African origin. She received 7 days of antibiotics for PROM and suspected sepsis but cultures remained negative. She had seizures at 9 days of age, and was initially treated with phenobarbitone. She had marked hypocalcemia, hypomagnesemia, and hyperphosphatemia and low circulating PTH concentrations (Fig. 1A), and additionally hypoglycemia and hypokalemia. There was no family history of hypocalcemia.
Before CSPI: She was commenced on alfacalcidol, calcium, magnesium supplementation ( After CSPI: At 11 weeks of age she started CSPI, and i.v. calcium infusion could be weaned and stopped at age 15 weeks. Seizures completely stopped. She was discharged for the first time in her life at the age of 6.5 months. Since then, she has required 3 admissions for hypocalcemia mostly due to intercurrent illness and vaccination, occasionally accompanied by tetany. Diazoxide could be reduced to 2 mg/kg/day but attempts to stop have so far been unsuccesful. She has not developed nephrocalcinosis and her renal function is normal. She has achieved normal motor and language milestones. She is growing normally.

Patient 6
Presentation: Patient 6 was born at 35 weeks gestation to parents of White European origin with a birth weight of 3.46 kg. Antenatal scans showed polyhydramnios. She had hypocalcemic seizures at 6 days of age, in association with hypomagnesemia and low circulating PTH concentrations. There was no family history of hypocalcemia.
Before CSPI: She was treated with alfacalcidol, calcium supplementation and magnesium supplementation. Because of insufficient seizure control she was commenced on PTH(1-34) bolus injections at age 18 months and continued on magnesium supplementation which ameliorated but not stopped her seizures. She required increased bolus doses of PTH(1-34) to maintain target calcium concentrations. She had polyuria, polydipsia and hypercalciuria related to Bartter syndrome type 5, but did not have evidence for nephrocalcinosis. She had mild speech delay but attends mainstream school.
After CSPI: At age 6 years she was started on CSPI, with initial improvement and halved PTH dose (from 3.6 µg/kg/day to 1.4 µg/kg/day) and she has not had any further seizures. One year into CSPI she required multiple admissions due to episodes of hyper-and hypocalcemia and increased PTH doses up to 3.4 µg/kg/day. In view of suspected tachyphylaxis she was weaned of CSPI and re-started on alfacalcidol and calcium supplementation. This was supplemented with PTH(1-34) bolus injections at times of parental concern of hypocalcemia, which resulted in hypercalcemia (serum calcium 4.25 mmol/L) (Fig S3), and this necessitated emergency hospital admission. The tachyphylaxis could potentially have been due to neutralizing antibodies to PTH(1-34), which were not measured. However, the hypercalcemia resulting from over-treatment from PTH(1-34), suggests such neutralizing antibodies were unlikely to be present at high concentrations.
She re-commenced CSPI two months later due to severe episodes of hypocalcemia and deterioration of her renal function. Her calcium concentrations have been erratic and she has needed several admissions for hypo-and hypercalcemia in combination with hyper-and hypovolemia related to her Bartter syndrome type 5. At age 11 yrs she experienced left hip pain and was diagnosed with left slipped upper femoral epiphysis and underwent bilateral epiphyseal pinning. At the same age she was restarted on oral medication (cholecalciferol, alfacalcidol and clacium supplements) with the aim to wean CSPI due to difficult management, and worsening of renal insufficiency. Ultrasonography did not reveal evidence of nephrocalcinosis, but she has CKD stage 5 with a severely reduced GFR of 12 ml/min/1.73m 2 , and is awaiting dialysis and renal transplantation. She has normal learning abilities, and growth is within the normal range.

Administration of PTH(1-34) by continuous subcutaneous infusion
PTH(1-34) (teriparatide) was administered using Medtronic Veo pumps in five patients Alfacalcidol was stopped at the start of infusion and calcium supplementation was weaned and stopped during the first week. The reservoir and infusion sets were changed every 3 days, as with DAPI (Thermo Fisher Scientific), and visualised on an Eclipse E400 fluorescence microscope, as previously described 5 . Protein expression was also confirmed using Western blot, as previously described 6 . CaSR-FLAG tag expression was detected using the HRP conjugated primary mouse anti-DDDDK antibody (AbCam, ab49763) at a 1:2000 dilution.

Luciferase reporter assays
Luciferase reporter assays were performed as described 7 . Briefly, 24 hours after transfection with WT or mutant CaSR constructs, cell growth media was replaced with starvation media

Statistical analysis
The statistical analysis plan did not include provision for correcting for multiplicity when conducting tests for secondary or other outcomes. Thus, the results of all analyses are reported as point estimates (arithmetic mean) with 95% confidence intervals. The widths of the confidence intervals have not been adjusted for multiplicity, therefore the intervals should not be used to infer definitive treatment effects for secondary outcomes. Biochemical data is biological replicates. Data analysis was performed in GraphPad Prism 8.    The solid and dotted lines represent the gender specific age-related BMAD reference intervals for a Hologic DXA scanner (mean, 5 th and 95 th centile) for 5-20 year olds 8 . BMAD reference intervals for children under the age of 5 years have not been established. Patients 1, 2 and 4 are represented in grey, blue and green. (Patient 1 BMAD 0.204g/cm 3 at age 3 years, patient 2 BMAD 0.167g/cm 3 at age 3.8 years, patient 4 BMAD 0.159 g/cm 3 at 3.6 years).