Mutation of the CYP2R1 Vitamin D 25-Hydroxylase in a Saudi Arabian Family with Severe Vitamin D Deficiency

Abstract

Context:

Inherited forms of vitamin D deficiency are rare causes of rickets and to date have been traced to mutations in three genes, VDR, encoding the 1α,25-dihydroxyvitamin D receptor, CYP27B1, encoding the vitamin D 1α-hydroxylase, and CYP2R1, encoding a microsomal vitamin D 25-hydroxylase.

Results:

Multiple mutations have been identified in VDR and CYP27B1 in patients with rickets, and thus, the roles of these two genes in vitamin D metabolism are unassailable. The case is less clear for CYP2R1, in which only a single mutation, L99P in exon 2 of the gene, has been identified in Nigerian families, and because multiple enzymes with vitamin D 25-hydroxylase activity have been identified. Here we report molecular genetic studies on two siblings from a Saudi family who presented with classic symptoms of vitamin D deficiency. The affected offspring inherited two different CYP2R1 mutations (367+1, G→A; 768, iT), which are predicted to specify null alleles.

Conclusion:

We conclude that CYP2R1 is a major vitamin D 25-hydroxylase that plays a fundamental role in activation of this essential vitamin.

Vitamin D deficiency remains the major cause of rickets among infants, children, and adolescents in countries throughout the world. A combination of nutritional, social, and climatic conditions explains much of this deficiency as illustrated in Saudi Arabia, a country otherwise rich in sunshine but in which rickets remains prevalent in different age groups due to limited sun exposure (1, 2). A recent retrospective study in 34 Saudi Arabian adolescents concluded that 58% were vitamin D deficient, 11.8% had low calcium, and the remaining 8.8% had potential genetic causes of rickets (3).

An understanding of vitamin D metabolism and action is necessary to diagnose genetic causes of inherited vitamin D deficiency. Unlike other fat-soluble vitamins, vitamin D is produced in mammals by a complex biosynthetic pathway involving nonenzymatic and enzymatic steps. In the first step of this pathway, a sterol precursor, 7-dehydrocholesterol, is converted by UV light into the secosteroid previtamin D. This intermediate spontaneously rearranges into vitamin D₃, which is then subject to 25-hydroxylation in the liver to generate the major circulating form of the vitamin, 25-hydroxyvitamin D₃. Several cytochrome P450 (CYP) enzymes with vitamin D₃ 25-hydroxylase activity have been identified, including CYP27A1 (4, 5), CYP2C11 (6), CYP2J3 (7), CYP2R1 (8), and CYP3A4 (9). It is not yet clear which of these P450 enzymes is the major vitamin D₃ 25-hydroxylase; however, loss of CYP2R1 was shown to cause selective 25-hydroxyvitamin D deficiency in several families from Nigeria (10–12). In contrast to the ambiguity regarding the enzyme responsible for hepatic 25-hydroxylation, abundant biochemical and genetic evidence indicates that CYP27B1 is the 1α-hydroxylase that forms the bioactive vitamin 1α,25-dihydroxyvitamin D (13, 14). This dihydroxylated secosteroid binds to the vitamin D receptor to mediate the endocrine actions of the vitamin in calcium and phosphorous metabolism (15, 16).

In the current study, we report two siblings from a Saudi family who presented with classical vitamin D deficiency and associated rickets. The condition arose in early childhood, improved with vitamin D therapy, but relapsed when vitamin D supplementation was stopped. This presentation suggested an underlying hereditary condition. Molecular analysis of genomic DNA revealed that both patients were compound heterozygotes for two previously undescribed mutations in the gene encoding the CYP2R1 vitamin D₃ 25-hydroxylase.

Patients and Methods

Patient 1

This individual is a 13-yr-old girl who first came to clinical attention at the age of 10 yr as a consequence of short stature. A history revealed an otherwise healthy girl whose birth weight had been normal as were other developmental milestones. Her nutritional history was uninformative with no features suggestive of malabsorption. Her main complaint was generalized bone pain and a limitation of physical activity. Examination revealed a well child whose height was below the 3rd percentile and whose weight was appropriate for height. Other than bowing of the lower limbs, obvious signs of rickets were absent. In the several years before visiting the clinic, she had taken different doses of vitamin D. Her initial laboratory results were indicative of classical vitamin D deficiency (Table 1).

Table 1.

Biochemical results (baseline and after therapy)

	Serum PTH (pmol/liter)	Serum total calcium (mmol/liter)	Serum phosphate (mmol/liter)	Serum alkaline phosphatase (U/liter)	Serum 25-OH-D (nmol/liter)	Serum 1,25(OH)2D (pmol/liter)
Normal value	1.6–7.2	2.20–2.70	0.7–1.52	<500 prepubertal, 40–150 in adults	50–125 (20)	41–127
Baseline results
Patient 1	46	1.4	1.4	445	<10	48
Patient 2	28	1.6	1.61	1145	<10	44
Results after treatment
Patient 1	5.7	2.3	1.58	288	23	50
Patient 2	16.7	1.8	1.6	293	28	46

All values were measured in serum.

Patient 2

This individual was the older brother of patient 1 and presented at age 14.7 yr. At age 5, he had developed severe progressive bowing of the lower limbs, generalized bone pain, and difficulty walking. He sought medical advice from multiple institutions, which concluded he was hypocalcemic. Vitamin D and calcium were supplied at different doses and time intervals. Before visiting our clinic, he had surgical correction of the lower limbs but still complained of bone pain and an inability to sustain average effort. His nutritional history was normal, and there were no symptoms of malabsorption. Examination revealed a generally well boy, with positive Chvostek sign, wide wrists, bowing of legs, and a waddling gait. His initial laboratory results confirmed a state of classical vitamin D deficiency (Table 1).

Biochemical measurements

Total serum 25-hydroxyvitamin D was measured by a competitive chemiluminescence immunoassay (DiaSorin LIAISON System, Saluggia, Italy). The limit of detection was 10–375 nmol/liter, with serum level below 50 nmol/liter defined as deficiency and a normal range of 50–125 nmol/liter. Serum PTH levels were measured using an immunoassay kit (Architect i2000; Abbott, Wiesbaden-Delkenheim, Germany). The limit of detection was 0.02–318 pmol/liter, with serum levels over 7.2 pmol/liter defined as abnormally high. Serum calcium, phosphorus, and alkaline phosphatase levels were measured using photometric assays (Architect i8000; Abbott).

Molecular biology

Genomic DNA was extracted from white blood cells using standard methods (16). Individual exons and their flanking sequences from the CYP2R1 gene were amplified by the PCR and subject to DNA sequence analysis as described previously (10).

Results

After evaluation, patient 1 was prescribed 5000 IU/d cholecalciferol and patient 2 was prescribed 10,000 IU/d. Patient 1 was compliant as reflected in her posttreatment laboratory results (Table 1), whereas patient 2 was less compliant and generated fluctuating laboratory results that included bouts of low calcium and 25-hydroxyvitamin D₃ throughout follow-up (Table 1). Symptoms of bone pain resolved with therapy in both patients.

The fact that serum levels of 25-hydroxyvitamin D3 remained low despite administration of supraphysiological doses of vitamin D₃ suggested that patients 1 and 2 might have selective 25-hydroxyvitamin D₃ deficiency. To test this hypothesis, genomic DNA was extracted from the white blood cells of both patients and their family members, and individual exons and their immediate 5′- and 3′-flanking sequences were amplified from the CYP2R1 gene. DNA sequence analysis revealed two previously undescribed mutations in the affected siblings and an inheritance pattern indicative of an autosomal recessive disorder (Fig. 1).

Fig. 1.

DNA sequence of CYP2R1 in patients 1 and 2 and pedigree of family. Affected individuals inherited one CYP2R1allele with a G to A transition in the splice donor sequence of intron 2 and a second allele with a T insertion in exon 3. The inheritance of these alleles in the family is shown.

Discussion

The siblings described here presented with typical clinical and biochemical features of classical vitamin D deficiency and associated rickets. The disease was evident based on history and abnormal laboratory values that included low serum calcium, high alkaline phosphatase activity, secondary hyperparathyroidism, and low 25-hydroxyvitamin D₃ levels but normal levels of 1,25-dihydroxyvitamin D. Therapy with supraphysiological levels of vitamin D₃ led to relief of symptoms and improvement of laboratory results, including serum levels of 25-hydroxyvitamin D₃, which were significantly low at baseline but rose to measurable levels with therapy, and normalization of secondary hyperparathyroidism.

The fact that serum 25-hydroxyviatmin D₃ levels rose upon treatment suggests either that the two CYP2R1 proteins encoded by the mutant alleles detected here have residual enzyme activity or that other vitamin D₃ 25-hydroxylases can contribute to the formation of 25-hydroxyvitamin D₃ when substrate levels are raised. Of these two interpretations, we favor the latter based on results obtained in mice from which the Cyp2r1 gene was deleted; these animals have no CYP2R1 enzyme activity but nevertheless have circulating 25-hydroxyvitamin D₃ levels that are 50% of normal (17). The identity of the pharmacologically relevant vitamin D₃ hydroxylase remains to be determined. The observed responses to treatment also suggest that the vitamin D receptor is functional in the two patients studied here.

The diagnosis of selective 25-hydroxyvitamin D deficiency was strongly suggested through molecular genetic analysis of CYP2R1, which revealed that patients 1 and 2 were compound heterozygotes for two previously undescribed mutations in the gene. One mutation, inherited from the father, was a G to A transition in the first nucleotide of the highly conserved splice donor sequence of intron 2, which is predicted to lead to improperly spliced mRNA that encode truncated CYP2R1 proteins or unstable mRNA. Although this mutation was not recreated in an expressible gene and studied by transfection, loss of this G in hundreds of other genes disrupts splicing and most likely does so in this case as well. The second mutation, inherited from the mother, was a T insertion in exon 3 that alters the normal translational reading frame and is again predicted to produce a truncated protein. A comparison of the truncated proteins specified by these alleles to the three-dimensional structure of the normal CYP2R1 enzyme (18) indicates that both would lack functional domains required for vitamin D₃ 25-hydroxylase activity.

With the L99P mutation described in the prismatic case of selective 25-hydroxyvitamin D deficiency (10), the present findings bring to three the number of CYP2R1 mutations in patients with this apparently very rare disorder. The L99P mutation was initially found in two affected brothers from a Nigerian family and was detected in heterozygous form in a screen of 100 random Nigerians, indicating a potential founder gene effect. In agreement with this notion, Levine et al. (11) reported the L99P mutation in two additional Nigerian families. The clinical findings in these Nigerian cases (19) were similar to the Saudi siblings described here and suggest that molecular analysis of CYP2R1 should be considered for individuals with an early childhood picture of chronic 25-hydroxyvitamin D₃ deficiency associated with rickets and who are otherwise healthy and have adequate vitamin D intake and/or sun exposure.