Biallelic and monoallelic pathogenic variants in CYP24A1 and SLC34A1 genes cause idiopathic infantile hypercalcemia

Objective Idiopathic infantile hypercalcemia (IIH) is a rare disorder of PTH-independent hypercalcemia. CYP24A1 and SLC34A1 gene mutations cause two forms of hereditary IIH. In this study, the clinical manifestations and molecular aspects of six new Chinese patients were investigated. Methods The clinical manifestations and laboratory study of six patients with idiopathic infantile hypercalcemia were analyzed retrospectively. Results Five of the patients were diagnosed with hypercalcemia, hypercalciuria, and bilateral medullary nephrocalcinosis. Their clinical symptoms and biochemical abnormalities improved after treatment. One patient presented at age 11 years old with arterial hypertension, hypercalciuria and nephrocalcinosis, but normal serum calcium. Gene analysis showed that two patients had compound heterozygous mutations of CYP24A1, one patient had a monoallelic CYP24A1 variant, and three patients had a monoallelic SLC34A1 variant. Four novel CYP24A1 variants (c.116G > C, c.287T > A, c.476G > A and c.1349T > C) and three novel SLC34A1 variants (c.1322 A > G, c.1697_1698insT and c.1726T > C) were found in these patients. Conclusions A monoallelic variant of CYP24A1 or SLC34A1 gene contributes to symptomatic hypercalcemia, hypercalciuria and nephrocalcinosis. Manifestations of IIH vary with onset age. Hypercalcemia may not necessarily present after infancy and IIH should be considered in patients with nephrolithiasis either in older children or adults.

1,25-dihydroxyvitamin D (1,25(OH) 2 D 3 ) by hydroxylation of the side chain.Loss of CYP24A1 function blocks catabolism of 25-hydroxyvitamin D (25(OH)D 3 ) and 1,25-dihydroxyvitamin D (1,25(OH) 2 D 3 ).Accumulation of these active forms of vitamin D3 enhances intestinal Ca absorption and bone reabsorption, resulting in hypercalcemia, hypercalciuria, nephrocalcinosis, and suppressed intact parathyroid hormone.In 2016, Schlingmann et al. [2] described another type of IIH (IIH type 2), which was caused by loss of function mutation of SLC34A1 and characterized by hypercalcemia, hypercalciuria, suppressed intact parathyroid hormone and hypophosphatemia.SLC34A1 gene encodes a member of the NaPi-II family (NaPi-IIa), which plays a central role in phosphate reabsorption in the proximal tubule by using the sodium-electrochemical gradient to drive phosphate translocation against its concentration gradient [3].SLC34A1 mutations cause NaPi-II loss of function, and result in renal malabsorption of phosphorus and suppressed FGF23.Hypophosphatemia together with decreased concentration of FGF23 leads to the stimulation of 1α-hydroxylase (CYP27B1) and inhibition of CYP24A1, that results in an increment of 1,25(OH) 2 D 3 levels, leading to hypercalcemia and hypercalciuria.
Both types of IIH present hypercalcemia, suppressed intact parathyroid hormone, hypercalciuria, and nephrocalcinosis.As nephrolithiasis is a common manifestation of IIH, IIH has been considered as a rare genetic cause of nephrolithiasis typically occurring in pediatric subjects with an estimated incidence of 1:33,000 live births [4].Without timely and effective diagnosis and treatment, chronic renal failure may develop [5].Only four cases have been reported in the Chinese region [6,7].Here we report six new Chinese patients with IIH, and describe the detailed clinical analysis, laboratory data collected, and the outcome after treatment.

Clinical description
This study was conducted in 6 IIH patients diagnosed between 1 month and 11 years of age at the Beijing Children's hospital of China from 2016 to 2022 (Table 1).Five patients (patient 1,2,3,5 and 6) were admitted with a suspected diagnosis of feeding difficulty, vomiting, poor weight gain, drowsiness and polyuria in infancy (Table 1).They all experienced symptoms and progressive exacerbation in early infancy.Among them, patient 5 was mistakenly applied cholecalciferol cholesterol emulsion (Vitamin D3 300000IU) at his 8 months old, which lead to a hypercalcemic crisis.
One girl (Patient 4) was diagnosed at 11 years with arterial hypertension and nephrocalcinosis.She presented feeding difficulty, vomiting, and slow weight gain at 2 months old, but she didn't test blood or urine electrolytes at that time.The symptoms improved spontaneously at 10 months old.When she was 6 years old, bilateral renal medullary calcification was determined by ultrasound.When she was 11 years old, she was referred for elevated blood pressure (130/80mmHg) and her creatinine clearance rate was at a lower limit (89.7 ml/(min*1.73m 2 ).
The clinical characteristics of the patients are displayed in Table 1, and Fig. 1 showed bone X-ray of the patients.

Treatment and follow-up
All patients stopped taking vitamin D and calcium preparations and avoided sunlight after discharge.The symptoms of patients 1, 2, 3 and 6 improved within a few days with hydration treatment, and the levels of serum calcium gradually decreased to normal (Fig. 2).Judging from the curves of serum calcium and urine calcium, the decrease of serum calcium levels is earlier than urine calcium, and patients still have persistent hypercalciuria when their blood calcium dropped to normal (Fig. 2).Hypercalcemic crisis in Patient 5 is difficult to be treated.His blood calcium fluctuates after hydration, furosemide, calcitonin and hemodialysis treatments.Finally, three months after diagnosis, his blood calcium decreased to normal after bisphosphonate therapy.
Among the 6 patients, patient 3 was lost to follow up, and the follow-up time for the other patients was 5 months to 2 years.During follow-up, Patient 1, 2, 5 and 6 remained asymptomatic with normal serum calcium level during follow-up, their physical and intellectual development were normal.Serum calcium level of Patient 4 remained normal during monitoring.However, her blood creatinine level is higher two years later, creatinine clearance rate declined to 77.7 ml/(min*1.73m 2 ).Her blood pressure fluctuated within the range of 120-130/75-80mmHg.

Discussion
In this article we report six Chinese patients with IIH, that were subjected to molecular and genetic analysis, and their IIH diagnosis was additionally verified by clinical and biochemical manifestations (Fig. 4).Among the six patients, five presented typical infantile hypercalcemia, while one patient (Patient 4) did not show hypercalcemia at diagnosis, but hypercalciuria was still obvious when she was 11 years old with a low-normal creatinine clearance rate.These data reflect the different manifestations of IIH at different life stages.Hypercalcemia can be temporary.The prognosis of hypercalciuria varies.In previous report, the hypercalciuria of IIH was difficult to improve [9].Our patient 4 still had hypercalciuria at the age of 11 years, however urinary calcium levels of our patient 2 gradually decrease to normal during follow-up.Besides the pathogenicity of gene mutations, IIH phenotype is also influenced by many environmental factors like diet, lifestyle, vitamin D intake, and activity of the  other vitamin D metabolism enzymes [10].Renal maturation may contribute to the onset of IIH, and haploinsufficiency is more obvious in infancy, suggesting that the occurrence of diseases caused by heterozygous variants is related to age [11].However, nephrocalcinosis is a consistent manifestation of IIH, as was previously reported [9].In patient 3, an ultrasound showed bilateral renal nephrocalcinosis at one-month-old, which occurred very early, probably before birth.In patient 4, nephrocalcinosis was first found at 6 years old and remained when she was 11 years old.There is insufficient evidence supporting that nephrocalcinosis tends into remission spontaneously with age.Renal calcification may be the only manifestation of the later stages of IIH [5].The GFR of patient 4 was lower than 90 ml/min/1.73m 2 .Janiec A et al. [5] showed that IIH patients have a greater risk of progressive chronic kidney disease, with a rate of 77%.The severity of the initial kidney injury rather than nephrocalcinosis appears to play a significant role as a trigger of progressive chronic kidney disease (CKD).Patient 4 presented feeding difficulty, vomiting and slow weight increase when she was 2 months old, however, GFR wasn't recorded.The other patients' GFR were low when admitted, and their GFR increased to normal range after hydration treatment.Renal function monitoring is needed in long-term follow-up.
In IIH patients, treatment included removing vitamin D supplementation, a low-Ca diet, and Pi supplementation in NaPi-IIa defect patients [12].Patients with hypercalcemic episodes may be treated with hydration and diuretics.If symptomatic hypercalcemia persists, bisphosphonates, calcitonin, glucocorticoids, and hemodialysis can be administered [13].In patients 1, 2, 3 and 6, hydration and diuretics reduced the blood calcium level to normal within a few days.Patient 5 still had persistent hypercalcemia, which could not be reduced to normal range after adding calcitonin or hemodialysis.Patient 5 began to receive routine vitamin D (500u/d) treatment after birth.He then gradually developed feeding difficulty and astriction, reflecting hypersensitivity of IIH patients to vitamin D supplementation.However, patient 5 was given oral administration of Cholecalciferol Cholesterol Emulsion (300,000 units of vitamin D3) at 8 months old without testing for vitamin D or serum calcium level.Following this treatment, his hypercalcemia symptoms aggravated and his 25(OH)D 3 level exceeded 400nmol/L, meeting the diagnostic criteria of intoxication by the Endocrine Society [14].Combined vitamin D intoxication aggravates treatment difficulties.We agree with the view that empirical therapy of vitamin D deficiency with high vitamin D doses is discouraged without previous documentation of 25(OH)D 3 concentrations and monitoring of 25(OH)D 3 and serum calcium levels [15].In the absence of detection, a single high dose IIH type 1and type 2 are described as an autosomal recessive disorder, however, individuals of a single heterozygote presenting chronic and latent symptoms have also been reported [16].In our patients, biallelic variants of CYP24A1 were found in two patients, while four were found to have mono mutant allele of CYP24A1 and/or SLC34A1.Functional validation tests are needed, however, clinical evidence helped confirm the disease.
In 2011, Schlingmann et al. [1] first reported biallelic mutations of CYP24A1 were agenetic cause of IIH.Cases with monoallelic variants in CYP24A1 gene were noticed and suggested an autosomal dominant inheritance with reduced penetrance [11,17].Here, our patient 1 with monoallelic CYP24A1 variant presented symptomatic hypercalcemia in infancy, providing a clinical reference for the view of the potential risk of developing hypercalcemia and related clinical manifestations if exposed to triggering factors [18].Unlike the cases of Molin A et al. [11] reported without renal disease, our patient 1 has significant renal calcification.In addition, the father of patient 5 carrying CYP24A1 mutation had kidney calculus, as well as his mother and sister.However, we cannot obtain DNA samples from the two women to verify whether their kidney calculus is related to the mutation.In a family survey by Brancatella A et al. [18], the rate of nephrolithiasis showed no difference between heterozygotes and the wild-type subjects, however, serum total calcium concentrations and 25(OH)D 3 concentrations were significantly higher in heterozygotes than in the wild-type subjects.These clinical cases reflect that monoallelic variants in CYP24A1 gene can cause infantile hypercalcemia, but the presence of renal calcification is still controversial.More clinical and laboratory evidence needs to summarize and the molecular mechanism needs to be explored.
The phenotype of IIH induced by SLC34A1 mutations (IIH type 2) was first described by Schlingmann et al. [2] in 2016, which is characterized by infancy onset with failure to thrive, polyuria, and medullary nephrocalcinosis.Hypercalcemia, suppressed PTH, hypophosphatemia, and impairment of renal phosphate conservation were demonstrated in laboratory data.IIH type 2 was also described as a recessive disease.Monoallelic heterozygous variants in SLC34A1 were first described to cause hypophosphatemic nephrolithiasis/osteoporosis-1 (NPHLOP1), Prie, D et al. [19] reported one patient and her only daughter showed symptoms and proposed the dominant inheritance of the disease.Schlingmann et al. [2] also described some heterozygous relatives of IIH type 2 patients having nephrolithiasis.However, no more clinical evidence was reported.Our patients with monoallelic SLC34A1 variant also presented with symptomatic hypercalcemia.Additionally, their parents carrying the SLC34A1 mutation were also found having nephrocalcinosis.Our reports provided clinical evidence of the effect of monoallelic heterozygous variants in SLC34A1.Increased urinary phosphate and calcium excretion, elevated plasma 1,25(OH) 2 D 3 and urolithiasis were shown in heterozygous SLC34A1-deficient mice (SLC34A1+/-), indicating the dominant negative effect of the mutant SLC34A1 protein on the function of the wildtype [19].The underlying mechanism of the dominantnegative effect needs further exploration.Patient 6 and his father were found carrying heterozygous c.1726T > C in SLC34A1 gene and heterozygous c.376C > T in CYP24A1 gene.Combined with the patient's hypercalcemia and hypophosphatemia, heterozygous c.1726T > C in SLC34A1 is considered responsible for IIH.Heterozygous c.376C > T in CYP24A1 gene might contribute to the severe phenotype of both patients.
Although the reported data are not sufficient for a final evaluation of the genetic mode of CYP24A1 and SLC34A1-related hypercalcemia, clinical evaluation and long-term observation are important for patients carrying monoallelic variants.Only four IIH patients have been reported in the Chinese population [6,7], who were identified with compound heterozygous mutations, without case report of monoallelic variants.Gene reports tend to ignore heterozygous variations.The frequency of kidney stones due to CYP24A1 deficiency was estimated between 420 and 1960 per 10,000 [20].According to Expert Consensus on Clinical Application of Vitamin A and Vitamin D in Chinese Children, vitamin D 400-800IU per day is routinely supplemented after birth [21].If we disregarded CYP24A1 variant carriers, a regular dose of vitamin D supplementation may promote the formation of renal medullary calcification.Investigation of CYP24A1 and SLC34A1 mutation frequency in the Chinese population and monitoring of blood calcium and urinary calcium during routine vitamin D supplementation need to be further explored.
Here, we described the clinical, biochemical, and genetic manifestations of six Chinese patients with IIH.Our study has certain limitations.Our study extended over a short period of time and included a limited sample size.Therefore, a cohort study with a long-term outcome of a larger sample size is needed in the future.
In conclusion, manifestations of IIH were different with age.Hypercalcemia and hypercalciuria can be gradually relieved, but nephrocalcinosis starts early and persists.Transient high calcium symptoms in infancy may go unnoticed in adult patients with nephrolithiasis.In addition, our reports provided some clinical evidence of the pathogenicity of monoallelic heterozygous variants in CYP24A1 and SLC34A1, suggesting that the monoallelic heterozygous SLC34A1 or CYP24A1 variant also contributes to symptomatic IIH.
patients underwent next-generation sequencing.Mutations in CYP24A1 or SLC34A1 gene were identified in all patients, combined with clinical manifestations, conforming to the diagnosis of IIH.The genetic test results of the patients and the verification results of their parents are shown in Fig. 3. Patient 4 and Patient 5 were found have biallelic mutations of CYP24A1.Patient 1 was found have a heterozygous CYP24A1 mutation (c.116G > C) and patient 2 and patient 3 each have a heterozygous SLC34A1 mutation (c.1322A > G and c.1697_1698insT, respectively).Patient 6 simultaneously have a mutation of CYP24A1 and a mutation of SLC34A1, and both mutations originated from his father.Parents' carriers of patient 2,3 and 6 have renal calcification.Six CYP24A1 variants (c.116G > C, c.287T > A, c.376 C > T, c.476G > A, c.823T > C, and c.1349T > C) and three SLC34A1 variants (c.1322A > G, c.1697_1698insT and c.1726T > C) were found.CYP24A1 c.823T > C (p.W275R) and c.376 C > T (p.P126S) were previously reported in patients with hypercalcemia[8].The other four CYP24A1 mutations and three SLC34A1 mutations have not been reported before.According to Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology, CYP24A1 c.1349T > C (p.F450S), c.476G > C (p.R159P) and SLC34A1 c.1322A > G (p.Y441C), c.1726T > C (p.W576R) are of uncertain significance (PM2 + PP3), with extremely low frequency and were predicted as "damaging" to the protein by SIFT, PolyPhen_2, Muta-tionTaster, GERP + + and REVEL.CYP24A1 c.287T > A (p.I96N) was reported with MAF (Minimum allele frequency) of 0.0017 (0.17%) in East Asians, and is predicted as "damaging" with software evidence of conservation and protein structure prediction (PP3).CYP24A1 c.116G > C (p.R39P) was predicted as PM2 + BP4.SLC34A1 c.1697_1698insT (p.G567Rfs*38) is located within exon 13, which causes a gene frameshift and results in a premature termination codon.

Fig. 1 X
Fig. 1 X-ray examination of limbs.(A) Long bone panorama of Patient 3 showed dense transverse banded shadows in Bilateral distal femur and tibial metaphysis.(B) Radiography of Patient 5 showed temporary calcification zone is slightly narrow and dense of in the metaphysis of the bone

Fig. 3 Fig. 2
Fig. 3 Pedigree of the patient's family.The affected patients are shown as a filled square

Fig. 4
Fig. 4 Diagnosis flow chart of hypercalcemia.The black line represents the diagnosis process of hypercalcemia in general, and the red line represents the diagnosis process of patients in this study.As PTHrP cannot be tested in China at present, and some patients do not have record of 1,25(OH) 2 D 3 level, assistance is given according to the medical history and related biochemical indicators and imaging examination to exclude other diseases

Table 1
Clinical and biochemical features of the six patients