Clinical And Genetic Analysis Of Two Chinese Families With Vitamin D-dependent Rickets Type Ia

Objiective:Vitamin D-dependent rickets type IA（VDDR-IA） is a rare autosomal recessive disorder, patients show the early onset of severe rickets characterized by hypotonia, muscle weakness, hypocalcemia and bone damage. It is caused by mutations in the CYP27B1 gene, which leads to 25-hydroxyvitamin D3 [25-（OH）D3] la-hydroxylase deficiency in the epithelial cells of the renal tube and decreased production of 1,25-dihydroxyvitamin D3 [1,25-（OH）2D3]. So far, 10 patients have been diagnosed with VDDR-IA by CYP27B1 gene mutation analysis in China. There has no genetic sequence analysis research about the complete pedigree. Reports about long-term treatment and follow-up of patients are still limited. The aims of this study were to analyze the clinical characteristics and individual therapy of two patients with VDDR-IA from two separate Chinese families, investigate the CYP27B1 gene in two large families, which help clinical diagnosis and treatment provide genetic counseling, and enrich the Chinese cases gene mutations database.Subject:We collected 2 patients and 17 members from two VDDR-IA families, as well as 101 healthy volunteers as control. Informed consent was obtained from all individuals before participating in the study. All blood samples are from the Fuzhou Children’s Hospital of Fujian.All individuals have not performed any genetic tests before.Methods:1. Clinical data and methods 1.1 Clinical data collection: medical history, physical examination, radiological films and laboratory examinations were investigated from 2 patients with VDDR-IA and their family members. 1.2 Serum 1,25-（OH）2D3 detection :Serum 1,25-（OH）2D3 of two patients and their family members were measured by ELISA method.2. CYP27B1 gene sequencing analysis 2.1 Genomic DNA extraction: Peripheral venous blood samples were collected and whole blood genome DNA were extracted by TIANGEN blood genomic DNA extraction kit. 2.2 PCR amplification: Refer to previous literature, the primers of all 9 exons and exon-intron boundaries of CYPZ7B1 gene were verified by Oligo 6 software. PCR amplification conditions were determined by preliminary experiments. 2.3 Detecting PCR products: PCR products were detected by 1.5% agarose electrophoresis and were sequenced by company.Result:1. Clinical data: Two patients had typical manifestations and radiological evidence of rickets. Laboratory data showed hypocalcaemia, hypophosphataemia, high levels of serum alkaline phosphatase, parathyroid hormone and 25-（OH）D3, and low serum 1,25-（OH）2D3 level. Laboratory data of their asymptomatic family members were normal. 2. Results of sequencing: Genetic sequence identified two patients were homozygous for a duplication mutation in exon 8 of CYP27B1 gene（1319₁325dup CCCACCC）. Their parents and some family members were carriers of the heterozygosis mutation. The mutation was not found in healthy individuals. 3. Results of patients’ treatment and follow-up: After treating with calcitriol and calcium, there were biochemical improvements with normalization of serum calcium and phosphorus, and radiographic evidences of compensatory skeletal mineralization.Conclusion:We describe two patients with typical clinical manifestations diagnosed as VDDR-IA confirmed by genetic sequence analysis, and both patients were homozygous for the same seven-nucleotide duplication of CYP27B1. This is the first report of homozygous patients of the seven-nucleotide duplication in Chinese patients.This report details the mutation of two large Chinese pedigrees from a small city, and compared the clinical characteristics and individual therapy of two patients. High-dose calcitriol was required and no long-term adverse effects were observed.