Letter to the editor regarding Duan et al, “CYP4A22 loss-of-function causes a new type of vitamin D–dependent rickets (VDDR1C)”

To the Editor:

We read with great interest the recent report by Duan et al¹ in the Journal of Bone and Mineral Research in which they proposed that a CYP4A22 variant that they identified is the basis for a new form of vitamin D–dependent rickets. As we detail below, the discordance between the typical clinical and biochemical features of vitamin D–dependent rickets and those of the patients described in this report, as well as the frequency of the described variant in the Asian population, leads us to reject the premise that this heterozygous CYP4A22 variant is a cause of vitamin D deficiency/dependency. Moreover, we submit that it is more likely that these 2 patients have hypophosphatemic rickets than vitamin D–dependent rickets. Below we provide the basis for our opinion.

The clinical features, particularly the dental abnormalities, are more suggestive of hypophosphatemic rickets than of vitamin D–deficiency/dependent rickets. In addition, the radiographs of the spine are more typical of hypophosphatemic rickets as well.

The biochemical features are not consistent with vitamin D deficiency/dependency. Specifically, the proband has a very low serum phosphate level with normal serum levels of PTH and calcium. In addition, as shown in the authors’ Supplemental Table 3, at baseline the proband’s 24-hour urine calcium excretion is 80 mg, which, although low, is higher than one would expect in severe vitamin D deficiency or dependency. The mother also has normal serum levels of calcium and very low serum phosphate levels, but levels of PTH and urinary calcium are not provided.

While it is true that both the proband and his mother have reduced serum concentrations of 25(OH)D, this is common worldwide. Moreover, the 25(OH)D concentrations are really only borderline low and, at least for the proband, are not low enough to cause secondary hyperparathyroidism. In vitamin D deficiency/dependency, low serum phosphate levels are the result of secondary hyperparathyroidism, which depresses the tubular reabsorption of phosphate (TRP). The TRP and/or tubular maximum phosphate reabsorption to glomerular filtration rate (TmP/GFR) are not presented for the 2 patients, but we would nevertheless predict that renal TRP is markedly depressed. Given that serum levels of PTH are not elevated, the authors should have considered other causes of renal phosphate wasting, such as excess circulating FGF23 or a primary defect in sodium-phosphate co-transporter expression or function. To wit, low serum concentrations of 25(OH)D are often present in patients who have X-linked hypophosphatemic rickets (OMIM 307800), likely due to coincidentally decreased vitamin D intake and/or excess CYP24A1 action.

The vitamin D challenge data are equally confusing. It is not clear why levels of 1,25(OH)₂D would increase after administration of 50 000 IU of vitamin D₃ but not after administration of 50 000 IU of vitamin D₂ when serum concentrations of substrates 25(OH)D₃ and 25(OH)D₂ showed equivalent increases. While the 25(OH)D increases after 50 000 IU of vitamin D are less than typically seen in normal subjects, the authors did not measure serum concentrations of parent vitamin D₂ or vitamin D₃, so interpretation of the response of CYP2R1 to vitamin D loading is uncertain.

The in vitro studies of Enhanced Green Fluorescent Protein (EGFP)-tagged CYP4A22 recombinant proteins are equally troublesome. There is very little expression of the wild-type protein and no expression of the mutant protein by immunoblot. The functional studies (the authors’ Figure 4) are difficult to interpret as it is not clear how much ethanol was added to the control wells and how much ethanol was added as diluent for cholecalciferol. The authors’ Figure 4 shows expression of CYP transcripts and proteins when cells are transfected with various vectors, with the greatest increase in CYP2R1 expression occurring in cells that had been transfected with the EGFP-tagged CYP4A22 cDNA, which is very strange given that little to no mutant protein is present. One significant problem with these studies is that HepG2 cells do not express high levels of CYP2R1, CYP27A1, or CYP27B1.

While we do not disagree that the CYP4A22 variant allele is likely to lack enzymatic function, we reject the idea that loss of 1 CYP4A22 allele would lead to a state of functional deficiency of the enzyme (ie, haploinsufficiency). Here, genetic studies provide compelling reasons why it is unlikely that heterozygous carriers of the CYP4A22 variant, c.900delG, p.Glu301ArgfsTer79, would have a rare form of rickets. First, this variant, which is more accurately annotated as c.901del, p.Glu301Argfs*80 (NM_001010969.4), is common. The variant has been overserved in 1976 patients in the heterozygous state from gnomAD v2, corresponding to a minor allele frequency (MAF) of 0.6997% and a maximum MAF of 4.094% in members of the East Asian population that includes the patients reported here (https://gnomad.broadinstitute.org/variant/1-47610223-TG-T?dataset=gnomad_r2_1). Second, the CYP4A22 gene has the lowest pLI score of 0, suggesting a good tolerance to heterozygous loss-of-function variants in the general population, further arguing against the causality of this variant in rickets. While it is possible that this variant might increase susceptibility to vitamin D–dependent rickets or hypophosphatemic rickets, a large cohort study would be necessary to investigate this hypothesis. Third, the CYP4A22 gene was not associated with serum levels of 25(OH)D in multiple published genomewide association studies. And fourth, mouse models in which Cyp4A14 alleles, the murine homolog of human CYP4A22, have shown disrupted develop hypertension^2,3 and high-fat-diet–induced obesity and insulin resistance.⁴ Not a skeletal dysplasia.

Based on the above, we do not believe that the authors have presented sufficient evidence to support their proposal that haploinsufficiency for CYP4A22 is a novel cause of vitamin D–dependent rickets. Further, we have strong reservations regarding the diagnosis of vitamin D–dependent rickets in these 2 patients and suggest that it is more likely that these patients have X-linked hypophosphatemic rickets owing to a mutation in PHEX that was not identified by the genetic testing scheme utilized by the authors.⁵

Funding

This work was funded in part by a grant from the National Institutes of Health (R01 DK112955) to Michael A. Levine.