Vitamin D-dependent rickets (VDDR) type 1: case series of two siblings with a CYP27B1 mutation and review of the literature

Abstract

Two siblings presented with clinical and biochemical features of rickets, initially suspected as hypophosphatemic rickets. There was no improvement initially, hence the siblings were reinvestigated and later diagnosed as having vitamin D-dependent rickets (VDDR) type 1 due to a rare mutation in the CYP27B1 gene encoding the 1α-hydroxylase enzyme. Both siblings improved with calcitriol supplementation. The initial presentation of VDDR is often confusing and algorithmic evaluation helps in diagnosis. We also present a brief review of the literature, including genetics.

Introduction

Vitamin D-dependent rickets type 1 (VDDR type 1) is a rare cause of refractory rickets, caused by mutation in the CYP27B1 gene. Children with mutations are normal at birth and present at around 6 months to 2 years of age with similar symptoms as nutritional vitamin D deficiency rickets. Common symptoms of the disease are delayed motor milestones, hypotonia, muscle weakness, and bone deformities, while some may present with hypocalcemic seizures in early infancy. Laboratory findings include hypocalcemia, hyperparathyroidism, low levels of 1,25(OH)₂D₃, and normal or high serum 25(OH)D₃ with radiographic features of rickets 1.

Case Presentation

Sibling 1. A 30-month-old male child presented with complaints slow growth, unable to stand, and bowing of legs. Six months prior to this presentation, he received vitamin D injection in another hospital, following which there was no improvement in symptoms. On physical examination there was hypotonia, frontal bossing, rachitic rosary, widening of wrist, and bilateral genu varum. Biochemical tests showed hypocalcaemia, hypophosphatemia, phosphaturia, and high serum ALP and PTH levels with normal vitamin D (Table 1). The possibility of renal tubular acidosis was ruled out in view of negative history of polyuria and normal pH in blood gas. Hypophosphatemic rickets was considered a possibility as fractional excretion of phosphate in urine was 48%, so he was started on phosphate, vitamin D, and calcium supplements, but there was no improvement. Further, differential diagnosis of VDDR type 1 was considered and the patient was started on calcitriol at 30 ng/kg/day along with phosphate supplementation (Joulie’s solution) at 2 mmols/kg/day. Both biochemical and clinical improvement was seen after starting treatment (Table 1).

Table 1. Parameters of the two siblings before and after treatment.

Parameters	Sibling 1		Sibling 2
	Before Treatment	After Treatment (15 months)	Before Treatment	After Treatment (15 months)
Weight (kg)	7.88	12.7	6	10.24
Height (cm)	72	89.5	69	83.5
Total calcium (mg/dL)	9 .2	9.9	8.8	9.9
Serum phosphate (mg/dL)	1.8	4.99	2.1	5.86
Alkaline phosphatase (IU/L)	1122	293	1238	318
25-OH vitamin D (ng/mL)	35.3	40.43	34.9	35.57
1,25-OH Vitamin D* (pg/mL)	-	77.2	-	97.4
PTH (pg/mL)	330.2	79.1	670.7	38.9
TRP (pg/mL)	52%		74.1%

PTH: Parathyroid Hormone; TRP: Tubular Reabsorption of Phosphate.

^*1,25-OH Vitamin D was done after treatment was started.

Sibling 2. This patient was the younger brother of case 1. He presented at the age of 15 months with similar complaints. On examination, he had wrist widening, large open anterior fontanelle, and bowed legs. Laboratory tests showed hypocalcemia, hypophosphatemia, phosphaturia, and raised serum ALP and PTH levels with normal vitamin D levels (Table 1). X-ray of bilateral wrists was suggestive of rickets. The patient was started on calcitriol at 30 ng/kg/day and phosphate supplements at 2 mmol/kg/day, which resulted in fast amelioration of both biochemical and clinical abnormalities. He was able to walk and his height increased at a speed of 10 cm/year. Treatment was adjusted in both patients as required according to the levels of serum calcium and phosphate, PTH, and urinary calcium/creatinine ratio during subsequent visits. The dose of calcitriol was gradually decreased to (20 ng/kg/day and 10 ng/kg/day) respectively in sibling 1 and 2 over few months of follow-up. Similarly, Joulie’s solution was given with a starting dose for low phosphate levels and was tapered and stopped during follow-up. Regular renal ultrasound was done on follow-up visits.

Gene sequence analysis showed a homozygous c.1294C>T (p.Arg432Cys) mutation in exon 8 of the CYP27B1 gene in both siblings. This mutation was previously reported in a patient with VDDR type 1, is pathogenic and functional assay revealed that this missense mutant retains only 6.9% of 1α-hydroxylase activity of the wild type2. The parents were non-consanguineous and asymptomatic. Although, unfortunately, genetic testing of parents was unavailable, it is predicted that both parents were heterozygous for the mutation. There was no pathogenic mutation in PHEX and FGF23.

Discussion

Vitamin D is an inactive pro-hormone and its activation requires sequential hydroxylation by cytochrome P-450 enzymes, resulting in formation of calcitriol or 1-alpha, 25-dihydroxyvitamin D₃ (1,25(OH)₂D₃). 25-hydroxy vitamin D 1-α hydroxylase is the rate-limiting enzyme in activation of vitamin D, primarily expressed in the kidney. In VDDR type 1, due to blockage of this enzyme activity, there is normal or elevated 25(OH)D₃ level, despite low or low-normal serum 1,25(OH)₂D₃, mimicking vitamin D deficiency clinically and radiologically3. In previous studies from India, VDDR type 1 as cause of refractory rickets has not been confirmed by genetic studies4 ^, 5. A differential diagnosis of VDDR type 1 should be done in patients not responding to vitamin D supplementation, once RTA is ruled out. In our patients, hereditary hypophosphatemic rickets was suspected initially in view of hypophosphatemia, phosphaturia, raised ALP, and normal vitamin D levels, but there was no improvement in the symptoms and serum ALP levels after starting vitamin D, phosphate, and calcium supplementation. Hence, a diagnosis of VDDR type 1 was considered, which was confirmed by identification of homozygous mutation in the CYP27B1 gene.

The plausible mechanism for phosphaturia in our index cases is secondary hyperparathyroidism. Fraser et al. in their original study showed that there are high circulating levels of PTH in stage 2 of VDDR6. PTH is a potent hormone that acts on G protein coupled PTHR1 receptor expressed on renal proximal and distal tubular cells, and decreases the phosphate reabsorption, hence causing renal leak of phosphate. This hypothesis is further supported by the fact that hypophosphatemia and phosphaturia were corrected by restoration of serum calcium levels using calcitriol and thereby suppression of hyperparathyroidism.

The CYP27B1 gene is located on chromosome 12q13.3, consisting of 17 helices, 6 β-strands, and 9 exons spanning 4859 bases7. Yamamoto et al. also identified amino acid residues whose point mutations cause conformational changes of protein, leading to absence of enzymatic activity. The amino acid R432, which was found to be mutated in our patients, is a part of the highly conserved ERR motif in helix K, and the ERR triad is proposed to form the conserved core folding in mitochondrial CYPs, so the mutation R432C may cause a significant disruption in the protein structure8.

To date, 71 different mutations in CYP27B1 have been reported in association with phenotypes of VDDR type 1 from different ethnic groups and different countries9. The reported mutations include missense mutations (most common, Table 2), deletions, insertions, nonsense mutations, splicing mutations, and so on1 ^, 10 ^- 12. The mutation seen in our patients was homozygous but the same mutation was previously reported as compound heterozygous mutations in a Chinese patient2. Few cases have been reported with normal concentrations of 25(OH)D₃ and 1,25(OH)₂D₃ and having homozygous missense mutations12, which were considered to cause conformational changes in protein, limiting the enzyme efficiency. This clinical heterogeneity suggests a genotype-phenotype relationship in this gene similar to other CYP genes. As our patient needed continuous supplementation of calcitriol, we believe that the mutation c.1294C>T (p.Arg432Cys) is probably associated with severe enzyme inactivity.

Table 2. Genotype phenotype correlation in different missense mutations of CYP27B1.

Genotype	Phenotype
Q65H	These mutations are associated with no enzymatic activity, leading to severe phenotypes.
R107H
P112 L
G125E
P143 L
D164N
S323Y
R335P
P382S
R389C
R389G
R389H
T409I
R429P
R453C
V478G
P497R
G57V	These mutations are associated with 5.6 to 12.1 % enzymatic activity, hence leading to moderate to severe phenotypes. However, clinical heterogeneity was found in patients having G57V mutation.
G73W
R459C
L333F
R432C
R492W
E189G	Partial 1-hydroxylase activity ranging from 2.3 to 22%.
E189K
L343F
T321R	Show no enzymatic activity but phenotype reported is mild.
G102E	This mutation has 80% reduction in enzymatic activity leading to severe phenotype but some phenotypes were reported to have mild disease.

Conclusion

Although a rare disorder, VDDR type 1 must be considered even in countries where vitamin D deficiency is common. Genetic analyses are beneficial for early diagnosis and further management of probable familial cases.

Glossary

Abbreviations

VDDR type 1

Vitamin D dependent rickets type 1

PHEX

phosphate regulating endopeptidase homolog X-linked

FGF23

Fibroblast growth factor 23

CYP27B1

Cytochrome P450 family 27 subfamily B member 1