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/1.73 m2. 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.

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)2D3 level, assistance is given according to the medical history and related biochemical indicators and imaging examination to exclude other diseases

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)D3 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)D3 concentrations and monitoring of 25(OH)D3 and serum calcium levels [15]. In the absence of detection, a single high dose of vitamin D given to patients with IIH can cause serious consequences. Due to renal calcification being a common manifestation of IIH, urinary ultrasound is a useful tool to document nephrocalcinosis and should be done before implementing such treatment.

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)D3 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)2D3 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 dominant-negative 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 un-noticed 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.