DOI 10.1515/jpem-2013-0183      J Pediatr Endocr Met 2014; 27(3-4): 335–341

Wei-Wei Hu, Yao-Hua Ke, Jin-Wei He, Wen-Zhen Fu, Chun Wang, Hao Zhang, Hua Yue, Jie-Mei Gu and Zhen-Lin Zhang*

A novel compound mutation of CYP27B1 in a Chinese family with vitamin D-dependent rickets type 1A Abstract Objectives: Mutations in the CYP27B1 gene, which encodes vitamin D 1α-hydroxylase, are the genetic basis of vitamin D-dependent rickets type 1A (VDDR1A, MIM 264700). The aim of this study was to investigate a novel CYP27B1 mutation and its clinical manifestations. Methods: VDDR1A was diagnosed based on clinical presentation, a physical examination, bone characteristics on an X-ray, and laboratory results. A molecular model of the CYP27B1 protein was constructed using the SWISS-MODEL server and Swiss-PdbViewer. Results: We sequenced the CYP27B1 gene in a 5-year-old male child who presented with growth retardation and a history of frequent hand, leg, and perioral twitching since the age of 12 months. We identified a compound heterozygous mutation consisting of two missense mutations: one in exon 7 (R389C [c.1165C > T]) and one in exon 8 (R459C [c.1375C > T]). We used the wild-type CYP27B1 as a receptor and calcidiol as a ligand to predict the interaction between the R459 site and calcidiol. According to the predicted structure, the wild-type R459 residue localizes to the pocket where CYP27B1 binds to its ligand. Conclusions: According to the Human Gene Mutation Database, the compound heterozygous mutation identified in our patient is novel and has not yet been reported in the literature. This mutation provides a new basis for further research on VDDR1A and for the development of clinical diagnostics. Keywords: CYP27B1 gene; mutation; vitamin D-dependent rickets type 1A. *Corresponding author: Zhen-Lin Zhang, MD, Metabolic Bone Disease and Genetic Research Unit, Department of Osteoporosis and Bone Disease, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, 600 Yi-Shan Rd., Shanghai 200233, P.R. China, Phone: +86-21-64369181-58442, Fax: +86-21-64081474, E-mail: [email protected]

Wei-Wei Hu, Yao-Hua Ke, Jin-Wei He, Wen-Zhen Fu, Chun Wang, Hao Zhang, Hua Yue and Jie-Mei Gu: Metabolic Bone Disease and Genetic Research Unit, the Department of Osteoporosis and Bone Disease, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, 600 Yi-Shan Rd., Shanghai 200233, P.R. China

Introduction Vitamin D exists in two forms: the D2 form is produced by plants, and the D3 form is produced by the action of ultraviolet radiation on 7-dehydrocholesterol in human skin. Vitamins D2 and D3 are biologically inactive prohormones that must undergo sequential hydroxylation before they can activate the vitamin D receptor. Both forms require a two-step hydroxylation at carbons 25 and 1 for activation. The first hydroxylation occurs in the liver, where vitamin D is hydroxylated to 25-hydroxyvitamin D3 [25(OH)D3] by hepatic 25-hydroxylase (1). The second step occurs mainly in the kidney; 25-hydroxyvitamin D is hydroxylated by the mitochondrial enzyme vitamin D 1α-hydroxylase to the biologically active hormone 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], which is, in turn, hydroxylated by the mitochondrial cytochrome P450 enzyme 25(OH)D-1αhydroxylase (1a-hydroxylase, P450c1α, CYP27B1) (1–3). 1,25-(OH)2D3 binds to the vitamin D receptor and plays critical roles in calcium and phosphate metabolism, bone growth, and cellular differentiation (2). Vitamin D 1α-hydroxylase deficiency, also referred to as vitamin D-dependent rickets type 1A (VDDR1A) or pseudovitamin D-deficiency rickets type 1A (PDDR1A), is an autosomal recessive disorder clinically characterized by hypotonia, muscle weakness, growth failure, hypocalcemic seizures in early infancy, and radiographic findings of rickets (3, 4). Previous genetic studies have revealed that VDDR1A arises from mutations in the CYP27B1 gene (4–6). To date, approximately 46 different CYP27B1 gene mutations have been identified, including 29 missense or nonsense mutations, 4 splicing mutations, 10 deletions, and 3 duplications (4, 6–11). Missense mutations are the

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336      Hu et al.: Mutation of CYP27B1 in rickets type 1A

I

II p.Arg459Cys

p.Arg389Cys

rickets and was treated with calcitriol (0.25 μg/day) and carbonate calcium (600 mg/day). After treatment, his cramps began to improve and his serum calcium levels increased. We noted bilateral bowing of his legs and the radiographic features of rickets, including splaying and threadlike shadows indicating growth plate calcification (Figure 2). The laboratory findings of the proband’s mother were as follows: serum calcium, 2.28 mmol/L; serum phosphate, 1.05 mmol/L; ALP, 76 U/L; PTH, 47.2 pg/L; and β-CTX, 429 ng/L. His father had the

A III

Figure 1 A genetic study of a family with pseudovitamin D ­deficiency rickets. The arrow indicates the proband.

most common type of CYP27B1 mutation, representing more than half of those reported. In this study, we investigated one individual with VDDR1A from a Chinese family and identified a novel compound heterozygous mutation consisting of two missense mutations located in exons 7 and 8 of CYP27B1.

B

Materials and methods Human subjects This study was approved by the Ethics Committee of the Shanghai Jiao Tong University Affiliated Sixth People’s Hospital. All the subjects signed informed consent documents before participating in the study. The proband with VDDR1A, his family (Figure 1), and the healthy control subjects were of Han ethnicity. The diagnosis of VDDR1A was made on the basis of clinical and radiographic examinations. The male proband was born by spontaneous delivery to a healthy, nonconsanguineous couple and was 5 years old at the time of diagnosis. He was asymptomatic at birth and displayed normal linear growth until the age of 12 months, when he began to display growth retardation and to frequently experience hand, leg, and perioral twitching. Beginning at 2  years of age, the proband exhibited leg bowing and delays in teething and growth. By the age of 4 years, the proband had lost 10 teeth. At a physical examination in 2011, he was 84 cm tall (  T]) and 8 (R459C [c.1375C > T]). Of all of the reported mutations in the 1α-hydroxylase gene, two are

the most common. The deletion of guanine 958 (958delG), as numbered from the CYP27B1 transcriptional start site (4), was found in 20 French Canadian alleles (10); a microsatellite haplotype analysis showed that these mutations arose from a single founder (10). In addition, a sevennucleotide insertion in exon 8 was found in 14 affected alleles (10, 18) arising from various ethnic groups with different microsatellite haplotypes (10). This Chinese family carried missense mutations in CYP27B1: R389C in exon 7 and R459C in exon 8. Missense mutations such as these are the most common type of CYP27B1 gene disruption, representing more than half of all reported CYP27B1 mutations. Missense mutations at amino acid position 389 have been found in individuals of different ethnicities (16, 17), indicating that this residue is

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340      Hu et al.: Mutation of CYP27B1 in rickets type 1A a hotspot for CYP27B1 mutations. R389C has been shown to cause a complete loss of 1α-hydroxylase activity (10). We used the wild-type CYP27B1 as the receptor and calcidiol as a ligand to observe the interaction between them. According to the predicted protein structure, the wildtype R459 residue is located in the pocket where ligand binding to CYP27B1 occurs (Figures 5B–D). The mutant R459C is only four residues away from a thiolate, cysteine 455, which is also highly conserved and responsible for heme binding (11). The R459 mutation might reduce enzymatic activity by a mechanism similar to the production of a cysteine thiolate 455. Clinical heterogeneity has been reported in PDDR1A patients. Wang et  al. (17) were the first to report on mild cases of PDDR1A; two patients had normal concentrations of 25(OH)D3 and 1,25(OH)2D3 and were homozygous for missense mutations. Furthermore, Cui et  al. (11) described six novel missense mutations in CYP27B1 that resulted in a partial loss of function. In our study, the proband presented with early disease onset, suggesting that he had a relatively progressive form of PDDR1A. He was a compound heterozygote for R389C and R459C. The clinical and genetic features presented by this patient were consistent with a lack of vitamin D 1α-hydroxylase (which is encoded by CYP27B1) and with a classic pattern of autosomal recessive inheritance. To date, nine Chinese patients with VDDR1A (including our patient) have been studied at the molecular genetic level, and ten distinct mutations have been identified (11). The mutation sites identified in the present work were identical to those reported by Cui et al.; R459C was a novel missense

mutation in the Chinese population in the latter study (11). The R459C mutation in exon 8 of the CYP27B1 gene results in an arginine-to-cysteine substitution, which is probably a hotspot in the Chinese Han population. We still need a future analysis of the phenotype–genotype relationship in Chinese PDDR patients with CYP27B1 gene mutation (19, 20). The problem of aligning, or establishing a correspondence between, residues of protein structures is fundamental in computational structural biology. Understanding protein function and protein evolution is a key aim in structural biology. Both are furthered by detecting all known protein structures that are geometrically similar to a given query structure. While there has been some encouraging progress, no types of protein spatial structure prediction method have had no deviation (21, 22). In summary, the compound heterozygous mutation described herein is a new mutation that has not previously been reported in the literature. The identification of this mutation provides a new basis for further research on VDDR1A and for the development of clinical diagnostics for this disease. Acknowledgments: This study was supported by the National Science Foundation of China (NSFC) (grant nos. 81370978, 81170803, 30800387, 81070692, and 81000360), STCSM10DZ1950100, and Academic Leaders in Health Sciences in Shanghai (XBR 2011014). Received May 12, 2013; accepted October 9, 2013; previously published online November 7, 2013

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A novel compound mutation of CYP27B1 in a Chinese family with vitamin D-dependent rickets type 1A.

Mutations in the CYP27B1 gene, which encodes vitamin D 1α-hydroxylase, are the genetic basis of vitamin D-dependent rickets type 1A (VDDR1A, MIM 26470...
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