nature publishing group Articles Translational Investigation Nutritional rickets: vitamin D, calcium, and the genetic make-up Mohamed El Kholy1, Heba Elsedfy1, Monica Fernández-Cancio2, Rasha Tarif Hamza1, Nermine Hussein Amr1, Alaa Youssef Ahmed1, Nadin Nabil Toaima1 and Laura Audí2 BACKGROUND: The prevalence of vitamin D (vitD) deficiency pigmentation are therefore main determinants of circulat- presenting as rickets is increasing worldwide. Insufficient sun ing vitD levels (2) that can be increased through dietary vitD exposure, vitD administration, and/or calcium intake are the intake (3). Interindividual differences in aspects of the vitD main causes. However, vitD system-related genes may also endocrine system have been well documented. They could be have a role. caused by genetic differences in important proteins in the vitD METHODS: Prospective study: 109 rachitic children com- endocrine system, such as VDR (4–6). pleted a 6-mo study period or until rachitic manifestations Multiple polymorphic variations exist in the VDR gene: disappeared. Thirty children were selected as controls. Clinical 5’-promoter variations can affect mRNA expression pat- and biochemical data were evaluated at baseline in patients terns and levels while 3’-UTR sequence variations can affect and controls and biochemistry re-evaluated at radiological mRNA stability and/or protein translation efficiency (7–10). healing. Therapy was stratified in three different protocols. Other genes encoding proteins in the vitD endocrine sys- Fifty-four single-nucleotide polymorphisms (SNPs) of five vitD tem (25-hydroxylase (CP2R1), 1-α-hydroxylase (CYP27B1), system genes (VDR, CP2R1, CYP27B1, CYP24A1, and GC) were 24-hydroxylase (CYP24A1), and vitamin D binding globulin genotyped and their association with clinical and biochemcial (GC)) also present polymorphic variations that could influ- data was analyzed. ence vitD synthesis efficiency and circulating 25-OH-D circu- RESULTS: Therapy response was similar in terms of radiologi- lating levels (9). cal healing although it was not so in terms of biochemical nor- It has long been speculated that genetic predisposition may malization. Only VDR gene (promoter, start-codon, and intronic influence the manifestations of vitD deficiency. Accordingly, in a genotypes) was rickets-associated in terms of serum 25-OH-D, prospective series of rickets patients, we attempted to analyze the calcium, radiological severity and time needed to heal. Eight role of genotypes of VDR and other genes related to the vitD sys- patients with sufficient calcium intake and 25-OH-D levels car- tem on rickets predisposition, severity, and response to therapy. ried a VDR genotype lacking minor allele homozygous geno- types at SNPs spread along the gene. METHODS CONCLUSION: Although patients presented epidemiologic Patients and Controls factors strongly contributing to rickets, genetic modulation Rachitic infants and children attending the Pediatric Outpatient affecting predisposition, severity, and clinical course is exerted, Clinic, Ain Shams University, Cairo, Egypt (latitude 30.01N) were included. Participation was voluntary and written informed con- at least in part, by VDR gene polymorphic variation. sent was obtained from parents or guardians. The study protocol was approved by the Ethics Committee of the University. This was a prospective study: 168 consecutive rachitic patients he prevalence of vitamin D (vitD) deficiency presenting as presenting within a time frame of 12 mo were included (recruitment period included winter and summer, as the sun is shining all the year Trickets in children is increasing worldwide owing to insuf- round in Egypt). Only 109 children either completed the 6-mo fol- ficient vitD intake and lack of exposure to sunlight. However, low-up period or continued on treatment until their rachitic manifes- considering that relatively few children with low serum tations disappeared. Inclusion criteria included clinical and radiological evidence of 25-hydroxyvitamin D (25-OH-D) levels have symptoms, rickets and age from 7 mo to 3 y. Children were excluded if they had genetic factors may additionally be linked to the manifestation taken vitD or calcium supplements during the 6 mo prior to the study, of vitD deficiency and rickets (1). if they had a history of kidney disease, tuberculosis, liver disease, pre- As natural food contains scant amounts of vitD, humans maturity, intestinal, cardiac, central nervous system, or other chronic disease. Bone disease (with the exclusion of rickets) or a history of naturally obtain vitD through precursor photosynthesis of hereditary forms of rickets together with weight <60% of the mean UVB-dependent vitamin D3. Sunlight exposure and skin weight for height were also exclusion criteria. 1Pediatrics Department, Ain Shams University, Cairo, Egypt; 2Pediatric Endocrinology Research Unit, VHIR (Vall d’Hebron Institut de Recerca), Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, CIBERER (Center for Biomedical Research on Rare Diseases), Instituto de Salud Carlos III, Barcelona, Spain. Correspondence: Laura Audí ([email protected]) Received 7 June 2016; accepted 23 August 2016; advance online publication 14 December 2016. doi:10.1038/pr.2016.222 356 Pediatric Research Volume 81 | Number 2 | February 2017 Copyright © 2017 International Pediatric Research Foundation, Inc. Rickets: nutrition and genetics Articles Thirty age- and sex-matched children were selected as controls Serum 25-OH-D was measured by an enzyme-linked immuno- from siblings of nonrachitic patients attending the outpatient clinic assay method (Immunodiagnostik AG, Bensheim, Germany). The or children with minor illnesses such as upper respiratory tract detection limit of the assay was 2.24 ng/ml. infections. Intact PTH was assayed by enzyme-linked immunoassay using Data Collection reagents supplied by Biosource (Biosource Europe SA, Nivelles, Belgium). Assay sensitivity was 2 pg/ml (range 15–1,040 pg/ml). Nutritional data collection included: type of feeding, whether breast In patients, 25-OH-D and intact PTH were measured in baseline fed or not, duration of breast feeding, and weaning. Dietary calcium samples and again at the end of the study (X-ray score of zero). In con- and caloric intake were calculated according to a food frequency trols, 25-OH-D and intact PTH were measured in baseline samples. questionnaire based on the pattern of diet in Egypt and compiled by the National Nutrition Institute (11). Calcium insufficiency was arbi- Radiological Evaluation trarily defined as an intake <50% of the recommended dietary allow- Assessment of rickets severity was made using standard X-rays of both ance (RDI) (12). Parents were asked to report the whole dietary intake wrists and knees. A 10-point scoring system was used (zero “normal”) in detail on the preceding day on two separate occasions. to a maximum score of 10 (19). Radiographs were examined by two Nutritional status was determined according to the Wellcome Trust independent observers and the score was taken as the mean of their Classification (13). scores. X-ray scores were determined at baseline, 2 wk after the start Sunlight exposure evaluation included children’s type of clothing of treatment and then monthly till radiological healing. The criterion and time spent outdoors every day. Sun exposure of 2 h per week for healing was an X-ray score of zero and time to healing was calcu- on the face and hands was considered sufficient to maintain normal lated as months needed to achieve a null X-ray score. 25-OH-D concentrations (14). The amount of sun exposure was mea- Vitamin D System Gene Polymorphisms sured by calculating the total body surface area exposed to the sun while wearing usual clothes using the Lund and Browder charts (15). Genotyping of 28 single-nucleotide polymorphisms (SNP) of VDR Exposure duration was calculated by finding the average time spent (Table 1), 4 SNPs of CP2R1 (25-hydroxylase: rs10741657, rs12794714, outdoors per week (in hours) during the period of direct sunlight rs10500804 and rs1993116), 1 SNP of CYP27B1 (1-α-hydroxylase: (0900–1500 h), where sun exposure index = hours of sun exposure rs4646536), 10 SNPs of CYP24A1 (24-hydroxylase: rs4809960, per week × fraction of body surface area exposed to sunlight (16). rs2181874, rs6022994, rs6022993, rs912505, rs751089, rs1570670, Assessment of socioeconomic status was done according to the cri- rs927651, rs927650, and rs4809957), and 11 SNPs of GC (vitamin D teria of El-Bohy (17), based on: paternal profession, educational level, binding globulin: rs222020, rs222016, rs1491719, rs4364228, rs4752, type of housing, family income, income source (regular or irregular). rs222035, rs7041, rs4588, rs2298850, rs3755967, and rs2282679), was performed using Applied Biosystems TaqMan and OpenArray tech- Patient Classifications and Therapeutic Groups nologies (Life Technologies, Carlsbad, CA). Patients were stratified from the start in three groups according to Statistical Analysis adequacy of sun exposure and calcium intake. The aim was to deter- mine whether by history alone, the cause of nutritional rickets could JMP 7.01 (SAS Institute, Cary, NC) was used for data entry and analy- be established and the appropriate treatment chosen. sis. Numeric variables were expressed as mean ± SD when normally Group 1: Thirty patients with adequate sun exposure (sun expo- distributed and as median and interquartile range when nonpara- sure ≥2 h/week) and inadequate calcium intake (<50% RDI).