Hereditary vitamin D-resistant rickets (HVDRR), also referred to as Hereditary vitamin D-dependent type II rickets (HVDDR-II), is a rare autosomal recessive disorder caused by molecular variation in the gene encoding the vitamin D receptor (VDR). HVDDR-II is characterized by early onset of rickets presentation and biochemical parameters such as low serum calcium levels, high parathyroid hormone, high alkaline phosphatase, and high 1,25(OH)₂D levels (1).

Several types of variants have been identified in the VDR gene as the leading cause of HVDDR-II, such as non-sense, substitutions, insertions, duplications, deletions, and splice-site (2). The primary defect caused by the mutant VDR gene is a decrease in intestinal calcium and phosphate absorption, which leads to decreased bone mineralization and rickets (3), despite high levels of 1,25(OH)₂D (4). Patients with HVDDR-II have rickets along with hypocalcemia, hypophosphatemia, secondary hyperparathyroidism, and elevated serum 1,25(OH)₂D levels, which is a hallmark for diagnosing this disease. The patient will present with the same characteristic features of nutritional rickets in the form of frontal bossing, bowing of the leg, growth failure, dental caries, and enamel hypoplasia (5).

The treatment of HVDDR-II is not standardized in most cases. Studies have shown that oral calcium supplements can improve patients’ clinical features. However, high doses are required to achieve therapeutic results. IV calcium is proven more potent and effective in improving clinical, radiological, and biochemical outcomes in a short period (2, 6–8). This can significantly improve the levels of calcium and phosphate in the first period of infusion, followed by improvement of alkaline phosphatase and parathyroid hormones.

This study presents the clinical, biochemical and genotype characteristics, long-term outcomes, and response to treatment in one of the most extensive patient series with HVDDR-II with variation in the VDR gene.

A total of the 42 patients with HVDDR-II were recruited, with females being 24 patients (57.1%) and males consisting of 18 (42.9%). The median age of patients was 12.5 years, with an IQR of [8-16]. Thus, the median age at presentation is 15.5 months, with an IQR of [7-38] ( Table 1 ). The most frequently reported symptoms are alopecia in 41 patients (97.6%), followed by short stature (height SDS<-2) in 36 patients (85.7%), bowing of legs in 9 patients (21.4%), delayed walking in 6 patients (14.3%), seizures in 4 patients (9.5%), and respiratory symptoms in 1 patient (2.4%) ( Table 2 ). A total of 30 patients (71.4%) presented due to a family history of HVDR Type II (one of the siblings), but still, the most presented after the first year of age.

As for initial laboratory tests, the PTH and Alkaline phosphatase (ALP) were high in all participants [IQR: 219-350 ng/L], [IQR: 646-1409 U/l], respectively. The levels of Ca and PO₄ were considerably low [IQR: 1.6-2.1 mmol/L, 0.8-1.3 mmol/L], correspondingly. However, all had high 1,25-(OH)2D3 levels [IQR: 432-479 pg/mL] ( Table 3 ).

Although 30 patients (71.4%) of patients had a positive family history of HVDDR-II, the majority 24 patients (78.6%) were diagnosed and treated after 12 months, and only 9 patients (21.4%) were diagnosed and started the treatment early (at 12 months of age and less). Furthermore, 35 patients (83.3%) were on IV calcium, and 7 patients (16.7%) were on oral calcium. In addition, 29 patients (82.9%) of 35 patients who received IV calcium required it every month or two, and 6 (17.1%) required IV calcium every three months. Consequently, the mean treatment duration for patients who completed the IV calcium treatment was 6.2 years ( Table 1 ).

The overall response to treatment was evaluated based on the growth parameters and the biochemical data. For the growth evaluation, the SDS height score was improved significantly (p-value: 0.0064) in the first 5 years of treatment, with a mean SDS score of -3.3 to -2.6 ( Table 4 ). Interestingly, when comparing the height SDS pre and post-treatment by gender, males reported a statistically significant improvement in height from (-4.0 to -2.3) (P=0.0000) compared to females (-3.1 to -2.8) (P=0.3612). Consequently, thirteen patients completed their growth and treatment with a mean height of 144.6 cm in females and 152.4 in males ( Table 5 ).

Regarding the biochemical data, the median levels of ALP, Ca, and PO₄ were considerably improved compared to the baseline, 929.5 to 315.5 U/l, 1.9 to 2.12 mmol/L, and 1.0 to 1.5 mmol/L, respectively. The median PTH levels improved from baseline 289 to 131 ng/L at the last follow-up but were still high (see Table 6 ). Nephrocalcinosis was absent in all patients, as evidenced by annual renal ultrasound scans. Additionally, the calcium-to-creatinine ratio, monitored every 3 months, consistently remained low across all patients.

Moreover, the radiological features before and after treatment are shown in ( Figure 1 ). After one year and a half on treatment (6 weeks IV calcium, then maintained on oral calcium 5 gm every 6 hours daily), the images showed interval improvement ( Figure 1B ) compared to the previously seen growth plate widening and metaphyseal irregularity/fraying ( Figure 1A ), with interval metaphysical sclerosis in keeping with healing rickets. All patients were confirmed genetically to have HVDDR-II.

The molecular genetic testing identified six different variants and all variants were Homozygous and already reported before shown in ( Table 7 ) as follows: c.885 C>A (p.Tyr295Ter) in 26 patients (61.9%), followed by c.88 C>T (p.Arg30Ter) (n=8; 19.0%), c.1036G>A (p.Val346Met) (n=4; 9.5%), c.820C>T (p.Arg274Cys) (n=2; 4.8%), c.803 T>C (p.Ile268Thr) (n=1; 2.4%), and c.2T>G (p.Met1?) (n=1; 2.4%). The molecular genetic testing identified known Homozygous variants in all patients ( Table 7 , Figure 2 ). The predicted effect of each variant identified in this cohort was found to be pathogenic/disease-causing using in silico prediction algorithms tools described in the methodology.

There was no genotype-phenotype correlation, but it was noticed that three out of four patients with the gene variant c.1036G>A (p.Val346Met) responded to relatively high doses of oral calcium compared to other gene variants whom mainly required IV calcium infusion for years. Interestingly, patients with the following variants demonstrated significant improvement in height with treatment: c.88C>T (p.Arg30Ter), c.885C>A (p.Tyr295Ter), and c.1036G>A (p.Val346Met) (P<0.05) (See Table 8 ). Nevertheless, there is a negative correlation between the gene variants and their growth response to therapy ( Table 8 ).

All our patients present with normal birth weight and length, but after the 1^st year of life, they develop growth failures. Our study showed that the two patients with the c.820C>T (p.Arg274Cys) pathogenic variant have normal height at presentation regardless of the age of presentation (see Table 8 ). All our patients had a permanent catheter for calcium infusion, with multiple catheter-related complications developed, such as central line blockage in 27 patients (64.3%), which required changing the porta catheter in most of them. Infection developed in 4 (9.5%) of the cases.

HVDDR-II is a rare autosomal recessive disease in the vitamin D receptor (VDR), with ∼ 100 cases reported worldwide. Most of the reported patients were from the Middle East (17). This article presents the largest cohort study of patients with HVDDR-II with typical clinical, biochemical, and radiological evidence. All of them had alopecia except for one case that shared the same pathogenic variant with another patient with alopecia. Alopecia in HVDDR-II is considered a sign of resistance in both hormonal and therapeutic response (18). In our study, patients continued to have total alopecia, unchanged during the follow-up period.

The growth charts of the patients who reached final height showed that the growth velocity improved with the onset of puberty ( Figure 3 ). However, male patients had more growth retardation at the onset of presentation in comparison to female patients (see Table 5 ).

The biochemical data at presentation showed that males had low serum calcium levels while females had serum calcium at a lower limit of normal range and elevated PTH levels. Although both genders had the same level of low phosphate and high ALP and marked elevation of 1-25(OH) 2D3 levels, there is a negative correlation between the gene pathogenic variant and abnormal biochemical data.

We found six different variants of VDR in our cohort, all of which have been reported before in patients from the Middle East ( Table 7 ) (11–16). The most common in our cohort was the c.885C>A (p.Tyr295Ter) variant, followed, in order of prevalence, by c.88C>T (p.Arg30Ter), c.1036G>A (p.Val346Met), c.820C>T (p.Arg274Cys), c.803T>C (p.Ile268Thr), and c.2T>G (p.Met1?).

Previous studies showed that the use of high vitamin D doses could improve the patient’s clinical presentation and biochemical parameters (18, 19). Regarding our study, all patients were given very high doses of vitamin D3 or vitamin D analogs for at least 3 months with no improvement prior to their referral to our hospital. Most of our patients required IV calcium infusion except 7 patients treated with high doses of oral calcium (5-10 grams per day) with good response. Three patients out of four with the (c.1036G>A; p.Val346Met) variant responded to oral calcium, while most patients with the (c.885 C>A; p.Tyr295Ter) variant required IV calcium management for a long duration, suggesting that the type of genetic pathogenic variant may guide the management of this condition.

Studies showed that their serum PTH and ALP levels returned to normal values in patients treated with IV calcium for 5 days monthly after 13 months of treatment. Serial X-ray evaluation demonstrated a dramatic healing of rickets (3). Another study reported that 5 out of 6 patients receiving IV calcium through a peripheral line once a week had improved clinical, biochemical, and radiological characteristics after 2.6 years (20). In our cohort, most patients required IV calcium for at least 5 years and then continued on high doses of oral calcium supplements. Most patients continued to have high PTH despite the improvement of other biochemical and radiological findings, even with frequent IV calcium infusions. All patients who improved and shifted to oral calcium didn’t require IV calcium again. The follow-up of our patients with a median duration of 10 years showed that patients responded very well to the treatment, and height measures improved.

Skeletal manifestation of HVDDR-II can happen due to the involvement of a direct action of vitamin D on the muscle through VDR (21). Furthermore, these manifestations may vary in presentation, and patients with mild defects can be identified during early childhood. The skeletal manifestations include metaphysical cupping and widening, flattening of the skull, dorsal kyphosis, bowing of the legs, and spontaneous remission of the rachitic feature, which can occur between 7 to 15 years when they reach puberty (22). Most of our patients with bone deformities at the onset of presentation improved after enhancing biochemical parameters. In our study, 4 patients out of 13 who have reached their final growth have a mild bowing of the femur, gene varus, and coxa vara, and only 1 patient required referral to orthopedic service for surgical intervention.

Alternative management of HVDDR-II with persistent hyperparathyroidism has been the administration of cinacalcet, a calcimimetic that activates the Ca sensor receptor, enhancing its sensitivity to circulating serum calcium concentrations and thereby decreasing PTH secretion; short term of 2 months administration of cinacalcet to 3 years old with HVDDR enabled relatively long term resolution of clinical, biochemical, and radiographic manifestations of this disease with maintenance therapy of IV calcium infusion (23). We are currently conducting a clinical trial on the use of cinacalcet in cases with HVDDR-II. During the treatment course, our patients were monitored for hypercalciuria and nephrocalcinosis via urinary calcium, creatinine, and renal ultrasound serially every 1-2 years, which showed no hypercalciuria nor nephrocalcinosis seen in any of our patients.

The current study reported valuable insights into VDDR II clinical features and management. However, its retrospective nature limits its generalizability. Therefore, prospective controlled studies are warranted to investigate treatment response in VDDR II patients better.

This is the largest cohort of patients with HVDDR-II, showing the clinical characteristics and response to current management with the challenges that show that there is still a need for better therapeutic approaches for this population, and these families need genetic counseling for the prevention of recurrence of the disease.

The data presented in the study are deposited in the ClinVar repository (https://www.ncbi.nlm.nih.gov/clinvar/), accession numbers: SCV005043784, SCV005043358, SCV005043357, SCV005043355, SCV005043353 and SCV005043351. Further inquiries can be directed to the corresponding author.

The studies involving humans were approved by The Office of Research Affairs in King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia (reference number: 2235518). The studies were conducted in accordance with the local legislation and institutional requirements. The human samples used in this study were acquired from a by- product of routine care or industry. Written informed consent for participation was not required from the participants or the participants’ legal guardians/next of kin in accordance with the national legislation and institutional requirements.

AA: Conceptualization, Writing – original draft. AA-A: Conceptualization, Writing – original draft. AB: Data curation, Writing – original draft. RA: Formal analysis, Methodology, Writing – original draft. FJ: Data curation, Writing – original draft. KR: Formal analysis, Visualization, Writing – review & editing. FI: Investigation, Methodology, Validation, Writing – review & editing.