Hereditary 1,25-dihydroxyvitamin D-resistant rickets (HVDRR): clinical heterogeneity and long-term efficacious management of eight patients from four unrelated Arab families with a loss of function VDR mutation

Introduction

Hereditary vitamin D-resistant rickets (HVDRR), also known as vitamin D-dependent rickets type II (VDDR2A, OMIM 277440), is a rare autosomal recessive disorder caused by pathogenic mutations in the vitamin D receptor (VDR) gene. Over 100 cases of HVDRR have been described [1]. The main clinical manifestations include an early onset of rickets, growth retardation, skeletal deformations and rachitic signs with or without alopecia. Affected subjects exhibit hypocalcemia due to impaired intestinal absorption of calcium, hypophosphatemia, secondary hyperparathyroidism and elevated circulating levels of 1,25-dihydroxyvitamin D, alkaline phosphatase and parathyroid hormone [1], [2], [3], [4]. Although patients show resistance to the 1,25-dihydroxyvitamin D treatment, calcium supplementation has been a recommended therapy [1], [2], [5].

Vitamin D exerts various biological effects on growth, bone formation, hair cycle, immune response and cancer [2], [3], [6], [7]. It plays a major role in bone mineral homeostasis to promote healthy growth and remodeling of bones. The actions of vitamin D are mediated through its circulating active form, 1,25-dihydroxyvitamin D, which is bound to the vitamin D-binding protein. After entering the cell, 1,25-dihydroxyvitamin D interacts with VDR, which heterodimerizes with the retinoid X receptor (RXR). The complex then binds to the vitamin D response elements in the promoter region of the target genes and recruits transcription co-regulators to control the gene expression [7], [8]. In addition to the canonical pathway, a number of other pathways of the action of vitamin D have been described [6], [7], [9].

Here, we describe eight new patients with HVDRR from four unrelated families with a loss of function mutation in the VDR gene. The clinical features of HVDRR patients and heterogeneity of manifestations are described. In addition, the effects of calcium supplementation in patients are presented with a focus on growth, clinical and laboratory findings. The study provides valuable information for the management of patients with severe HVDRR resulting from the loss of VDR.

Materials and methods

Patients

The eight patients belong to four unrelated families from the southern region of Saudi Arabia. Consanguineous marriages are shown in the family 1 pedigree and confirmed in other families by interviewing the parents of patients (Figure 1A). Informed consent was obtained from all individuals included in this study. The research related to human subjects has been complied with all the relevant national regulations, institutional policies and in accordance the tenets of the Helsinki Declaration, and has been approved by the authors’ Institutional Review Board or equivalent committee.

Figure 1:

Pedigrees, mutation analysis and clinical manifestations of the HVDRR patients.

(A) The eight patients (P1–P8) from four unrelated families are shown with filled symbols. Consanguineous marriages are depicted with horizontal double lines. The siblings of unknown gender or number, n, are shown with diamond symbols. (B) Total alopecia and frontal bossing in patient 5 (i); knee X-rays of patient 7 (ii) and patient 4 (iii) at the ages of 2 and 3 years, respectively; an X-ray of the legs of patient 4 at the age of 9 years showing bowing of the femora, coxa vara and genu valgum (iv); wrist X-rays of patient 7 (v) and patient 4 (vi) at the ages of 2 and 3 years show widening and fraying of the metatarsals; patient 8 at the age of 8 years, 1 year post-treatment, shows bowing of the tibia (vii). The fracture of the tibia is shown with an arrowhead. Radiographs of the wrists and knees show cupping and fraying of metaphyses, coarsening of the trabecular pattern and poorly ossified epiphyseal centers. (C) Sanger sequencing shows a homozygous AA mutation in a patient, a heterozygous CA in a carrier parent and homozygous CC in an unaffected normal individual (red arrows). The affected codon and amino acid sequences are shown below the chromatograms.

Results

Clinical presentations

On diagnosis, all patients showed growth retardation (Table 1, Figure 2). With the exception of an 8-month-old girl (patient 5) and a 7-year-old boy (patient 6), six of the eight patients weighed less than the average weight of Saudi children. The heights of seven patients were below the average height of Saudi children. Patient 5, who had an above average height at the age of 8 months, exhibited a reduced growth rate at 17 and 29 months. Because her elder sister was affected, she was tested and diagnosed with HVDRR. An early diagnosis and treatment helped her in attaining a normal growth rate as evidenced at 3 and 6 years of age (Figure 2). After the treatment, all patients showed a recovery of normal growth rates (Figure 2, Table 2).

Figure 2:

Heights of patients on diagnosis and after treatment.

For all patients, heights are plotted (black dots) on the background plots of normal growth during 2–20 years for girls (pink) and boys (blue) from Saudi Arabia. For patient 5, an additional chart of postnatal growth up to 36 months is shown. All patients showed normal growth rates after treatment.

Table 2:

Laboratory findings in cases at diagnosis and after treatment, current age and growth rates.

Data	Patient 1	Patient 2	Patient 3	Patient 4	Patient 5	Patient 6	Patient 7	Patient 8
Calcium, mmol/L
At diagnosis	2.05	1.70	1.82	1.63	2.04	1.40	1.61	1.86
1 year post-treatment	2.27	2.01	1.96	2.01	2.03	1.65	1.85	2.02
2 years post-treatment	2.30	nd	2.07	2.30	2.06	1.81	2.05	2.12
5 years post-treatment	2.34	2.37	2.26	2.45	2.25	1.79	2.11	2.13
10 years post-treatment	2.35	2.37	nd	nd	nd	nd	nd	2.20
Phosphate, mmol/L
At diagnosis	1.11	0.87	0.97	1.03	0.83	1.09	0.89	0.67
1 year post-treatment	1.36	1.01	1.02	1.23	0.80	1.23	0.92	0.79
2 years post-treatment	1.47	nd	1.09	1.49	1.00	1.39	1.14	0.87
5 years post-treatment	1.58	1.27	1.17	1.65	1.28	1.46	1.55	1.03
10 years post-treatment	1.60	1.31	nd	nd	nd	nd	nd	1.11
Alkaline phosphate, U/L
At diagnosis	533	593	1319	1575	638	567	1688	1990
1 year post-treatment	409	407	741	577	597	364	631	1386
2 years post-treatment	343	nd	527	286	463	330	440	1087
5 years post-treatment	265	253	250	188	238	277	239	570
10 years post-treatment	255	200	nd	nd	nd	nd	nd	197
Parathyroid hormone, ng/L
At diagnosis	310	138	226	370	160	69	226	668
1 year post-treatment	132	81	178	122	260	57	200	578
2 years post-treatment	118	nd	144	71	196	51	180	446
5 years post-treatment	50	40	77	36	152	42	69	263
10 years post-treatment	47	42	nd	nd	nd	nd	nd	122
Current age, years	15	21	8	11	6	14	7	15
Growth rates post diagnosis and treatment, cm/year
1st year	7	6	7.5	12.5	5.5	7	5	6
2nd year	9	nd	9	11.5	7.5	4	8.8	5
5th year	5.3	5.75	6.5	8.5	8	5.6	6.7	4.5
10th year	5.6	0.2	–	–	–	–	–	4.5

1. In healthy individuals, the normal concentration ranges are calcium 2.1–2.6 mmol/L, phosphate 1.0–1.5 mmol/L, alkaline phosphatase, 120–350 U/L and parathyroid hormone 15–65 ng/L. Concentrations in bold are abnormal out of the range values. nd means not determined.

Since birth, all patients exhibited total alopecia with the complete absence of hair from the scalp, eyebrows and eyelashes (Figure 1, Table 1). While alopecia was consistently present in all patients, there was heterogeneity of manifestations of other HVDRR-associated phenotypes. The varying phenotypes included frontal bossing in four of the eight patients, bowing of the legs in five patients, rachitic rosary in three patients and widening of both wrists in five patients (Table 1). Radiographs of the wrists and the legs show rickets with cupping and fraying of metaphyses, ossified epiphyseal centers and bowing of the tibia in affected patients (Figure 1B). In five of the eight patients, walking was delayed until 18–20 months of age, and tooth eruption was late in two patients. A fracture of the tibia was evident on an X-ray of Patient 8 at the age of 8 years (Figure 1B, panel vii). Patient 2 exhibited persistent bowing of the legs and was subjected to osteotomy on the tibia and fibulae at the age of 15 years. Figure 3 shows the improvement of bone deformities in the legs of a patient at 3 years after treatment.

Figure 3:

Effect of calcium treatment on bones.

Patient at the time of initial diagnosis (A) and after 3 years of treatment (B). The widening of the growth plate at the distal femur, improvement of the lateral bowing deformity of the tibia and femur are seen after the treatment. Before the treatment, mild osteopenia and bilateral coxa were present. After the treatment, the coxa vera of the proximal femur improved and there is a normalization of the neck-shaft angle to some degree. There is an overall improvement in the bowing angle of the long bones. The distal bowing of the tibia and fibula of the right leg shows an improvement of more than 17 degrees after 3 years of treatment.

In all patients, serum levels of 1,25-dihydroxyvitamin D were elevated to >430 pmol/L, which is well above the normal concentration range of 38–133 pmol/L (Table 1). In most of the patients, the serum concentrations of 25-hydroxyvitamin D were at the lower end of the normal range of 22–116 nmol/L. On diagnosis, patients exhibited hypocalcemia, hypophosphatemia and higher levels of alkaline phosphatase and parathyroid hormone. The laboratory findings in most of the patients improved to normal ranges after treatment (Table 2).

Ultrasonography of the kidneys of patient 4 at the age of 3 years showed grade 1 nephrocalcinosis bilaterally. The calcium treatment dose was not reduced. During the clinical visit at the age of 4 years, there was no sign of nephrocalcinosis. The other seven patients did not exhibit nephrocalcinosis.

Discussion

We describe eight new HVDRR patients from four unrelated consanguineous families with a recurrent c.885C>A (p.Y295*) loss of function mutation in the VDR gene who display heterogeneity of other HVDRR-associated manifestations. The patients were successfully treated with calcium infusion and monitored over a period of up to 11 years.

On diagnosis, while alopecia, hypocalcemia and increased levels of 1,25-dihydroxyvitamin D3, alkaline phosphatase and parathyroid hormone were found in all patients, there was heterogeneity of other phenotypic manifestations and in achieving developmental milestones. Though growth retardation was seen in most patients, frontal bossing, bowing of the legs, rachitic rosary and widening of wrists did not manifest in all patients. In five patients, walking occurred after 18 months and tooth eruption was delayed in only two patients. With the exception of an 8-month-old girl, patient age at diagnosis was not related to the variability of these manifestations. Although the 8-month-old patient did not exhibit frontal bossing, bowing of the legs, rachitic rosary and widening of wrists, her laboratory findings were consistent with HVDRR. Moreover, her above average height at the age of 8 months showed a reduced growth rate as seen by below average heights at 17 and 29 months of age. Because the HVDRR phenotypes may not appear at an early age, genetic testing of siblings of the patients affected with HVDRR is recommended. Despite having the same VDR mutation in all patients, the heterogeneity of phenotypic manifestations and developmental milestones could reflect influences of diet, lifestyle and other factors.

More than 50 mutations in the VDR gene have been described in HVDRR patients [1], [3], [11]. In HVDRR patients from the Middle Eastern and Egyptian origins, the truncating c.885C>A (p.Y295*) mutation has been described in addition to the p.R30*, p.G46D, p.D144N, p.R274H, p.R343C, p.R391H and p.R70Q mutations [3], [5], [12], [13], [14], [15], [16]. The c.885C>A (p.Y295*) mutation in eight patients in the present study has been described in other Arab patients (1, 12, 13, 16). The c.885C>A (p.Y295*) mutation might be specific to the Arab families where consanguineous marriages are frequent. Additional work is required and previously reported Arab families will be needed to investigate this hypothesis. Among the above VDR mutations, the c.885C>A (p.Y295*), p.R30*, p.R343C and p.R391H mutations produce total alopecia in HVDRR patients. It has been suggested that the VDR mutations affecting DNA binding or RXR heterodimerization, or those that result in the absence of VDR cause alopecia, whereas mutations affecting the VDR affinity for 1,25-dihydroxyvitamin D or coactivator interactions do not produce alopecia [2]. The persistent alopecia in our patients with the loss of function c.885C>A (p.Y295*) mutation corroborates with the fact that the hair growth cycle is dependent on the ligand-independent actions of VDR [2].

Clinical examinations and laboratory findings show that all patients responded to the treatment with elemental calcium. Patients exhibited normalization of the levels of calcium, phosphate, parathyroid hormone and alkaline phosphatase in the blood. Their growth rates were improved and rachitic symptoms were reduced. However, they continued to have total alopecia. Studies in the VDR knockout and hVDR-L233S mutant mice demonstrate that the action of VDR on hair growth is independent of the 1,25-dihydroxyvitamin D ligand [17], [18]. These studies and the persistent alopecia in patients in the present study despite the improvement in growth and other HVDDR phenotypes show that a VDR protein with or without a missense mutation affecting the 1,25-dihydroxyvitamin D ligand binding is essential for hair growth.

Although the VDR locus shows a linkage for idiopathic calcium nephrolithiasis (calcification in the lumen of the collecting system) [19], there is no clear evidence to implicate a defective VDR in nephrocalcinosis (deposition of calcium salts in the renal parenchyma). In a girl of Indian descent with HVDRR, mild nephrocalcinosis in the medullary region of both kidneys has been described [3]. She carried a 2-bp deletion in the 5′ donor splice site at the exon 3-intron boundary, causing exon 3 skipping and producing a premature truncation of the VDR protein with a complete loss of VDR function. In the present study, Patient 4, a 3-year-old boy with a complete loss of VDR function, also exhibited mild nephrocalcinosis that was resolved by the age of 4 years without reducing the dose of the calcium treatment. In another HVDRR patient with defective VDR, kidney stones were detected around puberty [20]. The nephrocalcinosis in HVDRR patients may have resulted due to the loss of functional VDR or treatment of patients with supraphysiological doses of elemental calcium.

An early diagnosis and treatment are important to prevent developmental effects of HVDRR. In the present study, Patient 5 was diagnosed at the age of 8 months due to genetic testing prompted by the fact that her older sister was affected by HVDRR. Upon early treatment, she exhibited normal growth without frontal bossing, bowing of the legs or other phenotypes caused by rickets. Her walking age and tooth eruption were unremarkable. This clinical observation emphasizes the importance of genetic testing for early diagnosis and long-term management of patients.

Conclusions

The study concludes that while the HVDRR patients with loss of function mutations can be managed with calcium treatment, the heterogeneous phenotypes seen in our patients with the same mutation are not reliable indicators of diagnosis or for assessing response to treatment. VDR sequencing and laboratory tests must be conducted to confirm a HVDRR diagnosis and to assess the effectiveness of the treatment.