Evaluation of the effectiveness of prophylactic oral vitamin D (cholecalciferol) in children with sickle cell disease

Journal Pre-proof Evaluation of the effectiveness of prophylactic oral vitamin D (cholecalciferol) in children with sickle cell disease

Carmen Garrido, Eduardo J. Bardón-Cancho, Verónica de los Ángeles Fajardo-Sánchez, María Elena Cascón-Pérez-Teijón, Marina García-Morín, Elena Cela, VIT-SICKLE Study Group, Cristina Beléndez, Cristina Mata-Fernández, Jorge HuertaAragonés, Laura Escobar-Fernández, Cristina Béliz-Mendiolaw PII:

S8756-3282(20)30008-9

DOI:

https://doi.org/10.1016/j.bone.2020.115228

Reference:

BON 115228

To appear in:

Bone

Received date:

12 September 2019

Revised date:

23 December 2019

Accepted date:

8 January 2020

Please cite this article as: C. Garrido, E.J. Bardón-Cancho, V. de los Ángeles FajardoSánchez, et al., Evaluation of the effectiveness of prophylactic oral vitamin D (cholecalciferol) in children with sickle cell disease, Bone(2020), https://doi.org/10.1016/ j.bone.2020.115228

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© 2020 Published by Elsevier.

Journal Pre-proof TITLE PAGE Title: Evaluation of the effectiveness of prophylactic oral vitamin D (cholecalciferol) in children with sickle cell disease Authors: Carmen Garridoa, MD, PhD, Eduardo J. Bardón-Canchob, MD, Verónica de los Ángeles Fajardo-Sánchezc, MS, María Elena Cascón-Pérez-Teijónd, MD, PhD, Marina García-Morínb, MD, Elena Celaa, MD, PhD, on behalf of VIT-SICKLE Study Group*.

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Affiliations:

Pediatric Hematology Unit, “Hospital General Universitario Gregorio Marañón”.

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Facultad de Medicina, Universidad Complutense de Madrid, España. Instituto

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Investigación Sanitaria Gregorio Marañón, Madrid, España. Instituto Nacional de Investigación Biomédica en Enfermedades Raras (CIBERER), Instituto de Salud Carlos b

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III, Madrid, España.

Pediatric Hematology Unit, “Hospital General Universitario Gregorio Marañón”.

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Facultad de Medicina, Universidad Complutense de Madrid, España. Instituto Investigación Sanitaria Gregorio Marañón, Madrid, España. Medical Student, “Hospital General Universitario Gregorio Marañón”. Facultad de

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Medicina, Universidad Complutense de Madrid, España. Musculoskeletal Section, Radiodiagnosis Department, “Hospital General Universitario

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Gregorio Marañón”. Profesora asociada Facultad de Medicina, Universidad Complutense de Madrid, España

∗A list of other contributors of the VIT-SICKLE Study Group with their affiliations is provided in the Collaborators section.

1. Carmen Garrido M.D., Ph.D. Pediatric Hematology Unit, “Hospital General Universitario Gregorio Marañón” 9 Maiquez Street 28007 Madrid, SPAIN E-mail address: [email protected] Phone number: 0034 915290037 Fax number: 0034 91529 0352 2. Eduardo J. Bardón-Cancho MD, Pediatric Hematology Unit, Pediatric Service, “Hospital General Universitario Gregorio Marañón”.

Journal Pre-proof 9 Maiquez Street 28007 Madrid, SPAIN E-mail address: [email protected] Phone number: 0034-677757377 / 0034-915290037 Fax number 0034-915290352. 3. Verónica de los Ángeles Fajardo-Sánchez M.S. Facultad de Medicina, Universidad Complutense de Madrid, España 4 Arzobispo Morcillo Street 28029 Madrid, SPAIN E-mail address: [email protected] Phone number: 0034 915290037 Fax number: 0034 91529 0352

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4. María Elena Cascón-Pérez-Teijón M.D., Ph.D. Musculoskeletal Section, Radiodiagnosis Department, “Hospital General Universitario Gregorio Marañón” 46, Dr. Esquerdo Street 28007 Madrid, SPAIN E-mail address: [email protected] Phone number: 0034 915290037 Fax number: 0034 91529 0352

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5. Marina García-Morín M.D. Pediatric Hematology Unit, “Hospital General Universitario Gregorio Marañón” C/ Maiquez, 9 28007 Madrid, SPAIN E-mail address: [email protected] Phone number: 0034 915290037 Fax number: 0034 91529 0352 6. Elena Cela M.D., Ph.D. Pediatric Hematology Unit, “Hospital General Universitario Gregorio Marañón” C/ Maiquez, 9 28007 Madrid, SPAIN E-mail address: [email protected] Phone number: 0034 915290037 Fax number: 0034 91529 0352 Address correspondence to: Eduardo Bardón-Cancho MD, Pediatric Hematology Unit, Pediatric Service, “Hospital General Universitario Gregorio Marañón”. 9 Maiquez Street, 28007 Madrid, SPAIN, E-mail address: [email protected] Phone number, 0034-677757377 / 0034-915290037 Fax number 0034-915290352. Word Count:

Journal Pre-proof a) Abstract: 249 words. b) Main Text (excludes title page, abstract, Conflicts of Interest, Acknowledgments, References, Tables, Figures, and Legends): 3731 words. Number of Tables, Figures, and Supporting Information files: 3 tables, 2 figures Short title: Prophylaxis with oral vitamin D in sickle cell disease Keywords: Sickle cell disease; cholecalciferol; vitamin D; prophylaxis; bone mineral density. Abbreviations key: Full term

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Abbreviation

25-hydroxyvitamin D

BMD

Bone mineral density

BMI

Body mass index

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25(OH)D

HDL-C HPCT

ISCD

Sickle Hemoglobin

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HbS

Bone densitometry

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DEXA

Reference centers, services and units

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CSUR

HDL cholesterol Hematopoietic progenitor cell transplantation International Society for Clinical Densitometry

IU

International units

OR

Odds ratio

SCD

Sickle Cell Disease

SEHOP

Spanish Society of Hematology and Pediatric Oncology

vitD3

Vitamin D3 or cholecalciferol

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Conflict of Interest statement There are no conflicts of interest to declare between the authors.

COLLABORATORS Collaborators of the VIT-SICKLE-Study Group: Cristina Beléndez M.D. Pediatric Hematology Unit, “Hospital General Universitario Gregorio Marañón”. Facultad de Medicina, Universidad Complutense de Madrid, España. Instituto

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Investigación Sanitaria Gregorio Marañón, Madrid, España. Instituto Nacional de

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Investigación Biomédica en Enfermedades Raras (CIBERER), Instituto de Salud Carlos

Cristina Mata-Fernández M.D., Ph.D.

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III, Madrid, España.

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Pediatric Hematology Unit, “Hospital General Universitario Gregorio Marañón”.

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Facultad de Medicina, Universidad Complutense de Madrid, España. Instituto

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Investigación Sanitaria Gregorio Marañón, Madrid, España.

Jorge Huerta-Aragonés M.D.

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Pediatric Hematology Unit, “Hospital General Universitario Gregorio Marañón”. Facultad de Medicina, Universidad Complutense de Madrid, España. Instituto Investigación Sanitaria Gregorio Marañón, Madrid, España.

Laura Escobar-Fernández M.D.

Pediatric Hematology Unit, “Hospital General Universitario Gregorio Marañón”. Facultad de Medicina, Universidad Complutense de Madrid, España. Instituto Investigación Sanitaria Gregorio Marañón, Madrid, España.

Cristina Béliz-Mendiola N.P. Pediatric Hematology Unit, “Hospital General Universitario Gregorio Marañón”. Facultad de Medicina, Universidad Complutense de Madrid, España. Instituto Investigación Sanitaria Gregorio Marañón, Madrid, España.

Journal Pre-proof Evaluation of the effectiveness of prophylactic oral vitamin D (cholecalciferol) in children with sickle cell disease ABSTRACT Background Vitamin D (25(OH)D) deficiency has become an emerging public health problem due to its influence on skeletal and extraskeletal diseases. Bone health in patients with sickle cell disease (SCD) is especially compromised and they are more likely to have

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25(OH)D deficiency than the general population. Despite this, there is little information

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on the efficacy of vitamin D3 (vitD3) prophylaxis and its role in improving bone

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mineral density (BMD) in this population.

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Procedures

A prospective, longitudinal, single-center study was conducted with 136 children with

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SCD monitored at a tertiary referral hospital for SCD. Demographic, clinical and

Results

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management data, 25(OH)D levels and bone densitometries (DXA) were collected.

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Eighty patients were included. There are significant differences between the means of each of 25(OH)D levels as a function of whether the patient started prophylactic treatment as an infant or not (35.71 vs. 27.89 ng/ml, respectively [p=0.014]). In multivariate analysis, 800 IU daily dose was shown as a protective factor (p=0.044) to reach optimal blood levels (≥30 ng/ml). According to Kaplan-Meier curves, patients younger than 10 years reached optimal levels earlier than older (p=0.002), as well as those who were not being treated with hydroxyurea (p=0.039). Conclusions VitD3 prophylaxis is a safe practice in SCD. It is important to start this prophylactic treatment when the child is an infant. The daily regimen with 800 IU could be more

Journal Pre-proof effective for reaching levels ≥30 ng/ml, and, especially in preadolescent and adolescent

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patients, we should raise awareness about the importance of good bone health.

Journal Pre-proof Evaluation of the effectiveness of prophylactic oral vitamin D (cholecalciferol) in children with sickle cell disease

Introduction Vitamin D (25-hydroxyvitamin D or 25(OH)D) deficiency has become a very prevalent condition in the general population1 and is an emerging public health problem receiving attention due to its influence on skeletal and extraskeletal diseases2. The first studies

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evaluating the effect of bone health on fractures in children were performed by

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Goulding et al.3. The use of vitamin D3 or cholecalciferol (vitD3) supplementation in

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otherwise healthy children with deficits in these nutrients has been demonstrated to

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significantly increase bone mineral density (BMD), thereby decreasing the risk of fractures4.

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Sickle cell disease (SCD) with autosomal recessive inheritance is the most prevalent

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genetic disorder identified in neonatal screening studies in several countries, including Spain5. Its primary characteristic is the presence of sickle hemoglobin (HbS) in

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erythrocytes6. Bone health in these patients is especially compromised and is characterized by complications in the form of dactylitis or other sickle cell (or vasoocclusive) bone crises, osteomyelitis, osteopenia or reduced BMD and osteoporosis secondary to chronic anemia and consequent bone marrow (BM) hyperplasia7. Patients with SCD are more likely to have 25(OH)D deficiency or insufficiency than the general population, and as much as 78% of the pediatric SCD population in countries with temperate climates is affected8. Some of the factors involved in this could be decreased vitamin D synthesis in their skin9, sun exposure, differences in their dietary habits10, and renal dysfunction. A cross-sectional study of 78 children with SCD at our center found that only 20.5% had concentrations of 25(OH)D > 30 ng/ml11, which was

Journal Pre-proof considered optimal for good bone health at the time of the study12. In addition to its connection to bone health, episodes of acute and chronic bone pain13,14, osteoporosis and osteonecrosis and infections15, 25(OH)D deficiency has also been associated with an increased frequency of cardiovascular disease16, asthma17 and nephropathy18. Despite the deficit of 25(OH)D in children with SCD, there is little information on the efficacy of vitD3 prophylaxis and its role in improving BMD in this

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population.

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Materials and methods

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The main objective of the study is to evaluate whether prophylactic vitD3 for children

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with SCD residing in the Community of Madrid achieves optimal levels of 25(OH)D and whether vitD3 supplementation is correlated with BMD, evaluated using bone

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densitometry (DXA)19,20.

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A prospective, longitudinal, single-center study was conducted with a cohort of 136 children with SCD monitored at a tertiary referral hospital for SCD (CSUR, for its

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initials in Spanish). Demographic, clinical and management data (sex, ethnicity, age, SCD genotype, type of vitD3 prophylaxis and duration of treatment, treatment with hydroxyurea, hypertransfusion regimen or hematopoietic progenitor cell transplantation [HPCT]), laboratory tests (last 4 serum 25(OH)D concentrations until December 31, 2017, which was the cut-off date for the inclusion of patients; the date on which optimal levels were reached was also recorded) and radiological findings (BMD Z-score, BMD in g/cm2 and percentage BMD gain or loss between the first DXA after the start of prophylaxis treatment and the most recent measurement before the completion of data collection) were collected.

Journal Pre-proof VitD3 prophylaxis was established as a routine practice at our center in 2012 based on the results of Garrido et al.11 and according to the recommendations of the Clinical Practice Guide for SCD from the Spanish Society of Pediatric Hematology and Oncology (SEHOP, for its initials in Spanish)21. VitD3 (cholecalciferol) supplements were administered on a daily (800 IU/day [12 drops]) or monthly (25,000 or 50,000 IU/month) schedule because several studies have shown that vitD3 has a certain superiority over vitamin D2 (ergocalciferol)22. As dictated by the universal

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recommendations, all patients born in Spain and who are undergoing regular follow-up

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by their local pediatrician, they start prophylaxis with 400UI/day of vitD3 from 15-30

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days of life until the 12-months of life. We will refer to them in this article as that they

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began prophylaxis in their infant period and joined the routine practice of our center after 12 months of life and not later, as in the other cases, which will be included in the

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group of those who did not start prophylaxis in the infant period. Adherence to

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prophylaxis was evaluated during follow-up visits, asking mainly the patient's parents

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for the medication that child was currently taking.

Inclusion criteria

- At least one consultation at the center before the inclusion cut-off date. - At least one determination of serum 25(OH)D concentration after the start of prophylactic vitD3 treatment. Exclusion criteria - Known growth or nutritional status disorders or chronic liver and/or kidney function disorders. - Treatment with drugs that affect the skeleton (except hydroxyurea; users were categorized as a separate population).

Journal Pre-proof - Non-adherence to prophylaxis during the study period. - Treatment with vitD3 after a diagnosis of osteopenia or osteoporosis. - Having reached adulthood and/or having died during the study period. Prior to inclusion in the study, all participants signed the corresponding informed consent form approved by the ethics committee of our center.

25(OH)D levels

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The 25(OH)D concentration was determined when blood was taken according to the

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protocol of care for SCD, which is every 3-12 months depending on the patient’s age,

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concomitant treatment and complications21. Therefore, for ethical reasons, it was not

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taken as an inclusion criteria to have a determination of 25(OH)D levels before starting prophylaxis, due to the significant number of patients who begin this prophylaxis in the

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first month of life. 25(OH)D levels were collected at least 6 months after the vitD3

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prophylaxis period began. Because 25(OH)D levels were made when the patient required a blood test for another reason, there is no exact range in months of levels

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among patients.

The concentrations were measured using the Liaison 25(OH) Vitamin D total assay (310-600), a direct, competitive chemiluminescent immunoassay. Our lab is governed by international standard ISO 9001, which includes ranges of normality, calibration and reproducibility. The reference ranges used in this study, although defined for the adult population, are also used in most SCD studies because there was no uniform consensus for this population at the time of study12. They are defined as follows: - Severe deficit: <10 ng/ml (25 nmol/l). - Moderate deficit: 10-19.9 ng/ml (25-49.9 nmol/l). - Insufficiency: 20-29.9 ng/ml (50-74.9 nmol/l).

Journal Pre-proof - Optimal levels: ≥ 30 ng/ml (75 nmol/l). DXA DXA was performed in children older than 3 years as this age is considered the limit for cooperation without the need for sedation. In order to draw inferences between 25(OH)D levels and BMD, according with the guidelines the first DXA of each patient was performed at least 6 months after the start of prophylaxis with vitD3, and therefore, of the first determination of collected 25(OH)D levels; and the second DXA after

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ultimate determination of collected 25(OH)D levels, in all cases. Measurements of the

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posteroanterior spine were performed; the hip and proximal femur were ignored given

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the great variability of these areas during growth, according to the recommendations of

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the International Society for Clinical Densitometry (ISCD) in children19. The equipment used in this study was the HOLOGIC ASY-00409 bone densitometer (Discovery

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Series), which fulfills the conditions for the study of pediatric patients. The results were

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interpreted by a radiologist specializing in densitometry and were expressed as BMD Zscores, which are based on the number of standard deviations of the BMD compared

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with other children of the same age, height, sex and ethnicity. The reference values used20 were as follows:

- Normal study: BMD Z-score > -2 SD. - Low BMD: BMD Z-score  -2 SD . BMD is not required for the diagnosis of osteoporosis in children with vertebral fracture. A BMD Z-score ≤ - 2DS indicates osteoporosis in children with a history of two or more long broken bones at the age of 10 years or three fractures of long bones at 18 years.

Statistical analysis

Journal Pre-proof Qualitative variables are expressed as absolute frequencies and percentages, and quantitative variables are summarized as measures of central tendency. The symmetry of the distributions was explored graphically with histograms and P-P plots. The association between quantitative variables was explored using Student’s t-test and ANOVA for parametric samples and Mann-Whitney U, Wilcoxon or Kruskal-Wallis U for nonparametric samples. The association between qualitative variables was analyzed using chi-square test, Fisher’s exact test or McNemar’s test as a function of the sample.

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Correlation studies were performed using the Pearson or Spearman correlation

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coefficient according to the sample distribution. The Kaplan-Meier survival test was

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used to analyze the time necessary to reach optimal levels of 25(OH)D. Likewise,

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simple and multiple regression studies were performed. Statistical analysis was

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performed using SPSS v. 25.0. Statistical significance was set at p < 0.05.

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Results

From the cohort of 136 patients with SCD, after review of the inclusion and exclusion

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criteria, a total of 80 patients were included. The majority of the excluded patients had lost follow-up during the study period, and adherence to prophylaxis with vitD3 was compromised, or not having 25(OH)D levels collected. The descriptive statistical results of the included population with respect to the epidemiological, analytical and radiological variables are shown in Table 1.

25(OH)D levels The frequencies of each of the reference ranges of the 25(OH)D concentrations measured are shown in Figure 1.

Journal Pre-proof There are significant differences between the means of each of the 25(OH)D concentration levels as a function of whether the patient started prophylactic treatment as an infant (Table 2). The monthly prophylaxis regimen between 25 000 IU and 50 000 IU was decided based on baseline 25(OH)D levels, so it has not been possible to analyze the efficacy of both regimens for the duration of this study. Differences (p < 0.001) between the most recent 25(OH)D levels were also observed depending on whether the patient had had a transplant (40.83 ng/ml vs. 30.66 ng/ml,

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respectively), although such differences were not observed for earlier 25(OH)D levels.

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The mean levels of 25(OH)D did not show significant differences in terms of sex,

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ethnicity, anemia genotype, prophylaxis regimen, treatment with hydroxyurea or

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hypertransfusional regimen, or in the BMD Z-scores from the initial and final DXA. There was a significant negative correlation between the different determinations of

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serum 25(OH)D with respect to the age of the patient (-0.471 for oldest 25(OH)D

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levels, p<0.001) and the age at the onset of prophylaxis with vitD3 (-0.485, p<0.001); this correlation was weakened at the most recent levels, i.e., with increased duration of

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prophylactic treatment (-0.250, p=0.025 and -0.237, p=0.035 respectively). In contrast, there was no significant correlation between 25(OH)D levels and the percentage of gross gain or loss of BMD, either annually or according to the BMD Z-score of the DXAs.

There was a negative correlation of the first 25(OH)D levels collected after prophylaxis began and the BMD in g/cm2 on both the first (-0.376, p=0.007) and final DXA (-0.412, p=0.001), although the differences became nonsignificant for the more recent (longer time on prophylactic treatment) measurement of 25(OH)D concentration (-0.124, p=0.381 and -0.237, p=0.059 respectively).

Journal Pre-proof Bone mineral density and BMD Z-scores There were differences when comparing the BMD Z-score and BMD values between DXAs, and the last DXA was associated with higher BMD Z-scores and BMD values than the initial DXA for 94.2% of cases (p < 0.001 in both cases). Differences were also observed between BMD values (for both the initial and the last DXA performed) and in annual BMD gain or loss (p = 0.019), depending on whether the patient had started vitD prophylaxis in infancy.

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The BMD values were correlated with patient age, with a coefficient of 0.672 (p <

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0.001) for the first DXA performed after the start of prophylaxis and a coefficient of

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0.784 (p <0.001) for the most recent DXA. In addition, a correlation of -0.319 was

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observed between age and the percentage of annual BMD gain (p = 0.024). In contrast, there was no significant correlation between the percentage of BMD gain or loss or its

Scope of optimal levels

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annual rate as a function of the duration of prophylaxis.

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The survival curves indicate significant differences in the time to reach 25(OH)D concentration levels considered to be optimal after the distribution of the variable was graphically explored and the cut-offs were defined as 10 years of age (p=0.002) and treatment with hydroxyurea (p=0.039). No differences were observed between a daily or monthly

prophylaxis

regimen

(Figure

2).

Journal Pre-proof Differences were also observed between the mean age of the patients and the age at which they started the vitD3 prophylaxis program according to whether they reached optimal levels of 25(OH)D or not (10.37 years vs. 14.31 years and 4.22 years vs. 9.05 years, respectively) [p = 0.002 and p < 0.001, respectively]. There was also a trend towards significance (p = 0.086) between the mean vitD3 prophylaxis times of the patients who reached optimal levels and those who did not (60.93 months vs. 50.19 months, respectively). There was also a difference in the time required to reach optimal

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levels depending on the monthly regimen used (34.97 months for 50,000 IU vs. 17.96

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months for 25,000 IU, p = 0.006).

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Differences were observed between the mean BMD in both DXAs performed as a

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function of whether optimal levels were reached (0.54 g/cm2 vs. 0.64 g/cm2, respectively, for the initial DXA [p = 0.001] and 0.59 g/cm2 vs. 0.77 g/cm2,

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respectively, for the most recent DXA [p = 0.044]). No significant differences were

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observed between the BMD Z-scores or the percentage of BMD gain or loss or its annual rate as a function of whether optimal levels were reached.

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The univariate and multivariate analyses for the calculation of odds ratio (OR) regarding the risk of not reaching optimal levels are shown in Table 3. No differences were observed in 25(OH)D levels, BMD Z-scores, BMD, percent BMD gain or loss or BMD annual rate, age at the start of prophylaxis or age of patients when comparing the two most commonly used prophylaxis regimens (800 IU/day vs. 25,000 IU per month), except in the case of the total time of prophylaxis at the end of the study (68.70 months vs. 55.63 months, respectively, p < 0.001). It was not necessary to suspend treatment due to toxicity for any patient. Bone fractures were not reported for any of the included patients.

Journal Pre-proof Discussion Deficiency of 25(OH)D is a common condition that is especially prevalent among individuals of African and African-American ethnicity23, and it has been considered a risk factor for acute and chronic pain24 and has been associated in multiple studies with cardiovascular, autoimmune and pulmonary diseases, as well as increased mortality25. A review of the prevalence of 25(OH)D deficiency in individuals with SCD shows that it ranges from 56% to 96%12 and is conditioned by the previously cited factors and by dysfunction,

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conversion

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renal

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1-alpha-25-

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BMD and an increased risk of bone fractures26,27.

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hydroxycholecalciferol, its active form. In addition, SCD is associated with a reduced

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The prevalence of 25(OH)D deficiency in our reference population was analyzed in a previous study and was close to 80%; 56.4% of the sample had levels below 20 ng/ml11.

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After these results were obtained, a vitD3 prophylaxis program was initiated. The

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prophylaxis regimen depended on factors such as expected adherence to treatment, patient age or the physician’s decision. The efficacy of this program was evaluated

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through the measurement of serum 25(OH)D concentration levels and the analysis of BMD by DXA. A total of 82.5% of the included patients achieved optimal serum 25(OH)D levels at some point in the follow-up, and in 64 patients (80%), at least one DXA was performed after prophylaxis began. The percentage of patients who reached optimal levels after the first 25(OH)D measurement increased from 50.0% to a maximum of 62.5%. Some of those who reached optimal levels during follow-up lost them again at some point, most likely due to poor adherence to treatment. Although the monthly dose of vitD3 (in the form of either 25,000 IU/month or 50,000 IU/month) was chosen to provide the greatest comfort to the patients and their families and to achieve the greatest compliance; the fact that the medication is not taken routinely (daily) can go

Journal Pre-proof against adherence to treatment. The monthly dose between 25 000 IU and 50 000 IU was decided based on baseline 25(OH)D levels, so it has not been possible to analyze the efficacy of both regimens for the duration of this study. A possible cause of lower 25(OH)D levels in these patients is a more severe SCD phenotype28, with an increased nutritional demand29 and a reduction in the absorption of nutrients due to damage to the intestinal mucosa, as previously described30. Han et al.25 recently reported that 25(OH)D levels are related to the expression of different genetic polymorphisms.

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Higher expression of SLC6A5 or CYP3A4 is associated with lower levels of 25(OH)D.

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Also associated with this is lower expression of the DBP and VDR genes, which,

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together with CYP3A4, are 3 genes involved in the metabolism of vitD3.

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Likely due to adherence to treatment, universal prophylaxis from birth to 12 months of age (with a globally agreed-upon regimen of 400 IU/day)22 and the continuation of

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prophylaxis with any regimen shows that patients with better levels of 25(OH)D reach

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older levels compared to other patients in any of the measurements that we took. We observed that patient age and the age at which prophylaxis was started, regardless of

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the regimen chosen, were negatively related to 25(OH)D levels, which confirms the importance of administering VitD3 supplements early. However, in the adult population, the exact opposite has been observed: higher levels of 25(OH)D are found as the age of the patient increases25. Regarding BMD and BMD Z-scores, in 94.2% of the cases in which two DXAs were performed, both values increased significantly. The increase in BMD could be because the patient was older at the second determination, which logically would be associated with an increased BMD. This is confirmed by the correlation coefficients obtained in our sample, but changes in the BMD Z-score could mean that the prophylaxis program is effective. A previous study conducted at our center31 with a smaller sample found no

Journal Pre-proof correlation between BMD Z-scores and 25(OH)D levels such as we found in the current study. In both studies, no differences were observed in the BMD Z-score between the group that received prophylaxis as infants and the group that did not; however, BMD was significantly higher in those who started prophylaxis in their first year of life. The Kaplan-Meier curves showed that patients younger than 10 years reached optimal blood levels of 25(OH)D earlier than others, which reinforces the importance of starting prophylaxis early. In addition, patients who were not undergoing hydroxyurea treatment

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achieved optimal 25(OH)D levels earlier. This could mean that more critical patients,

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who have an indication to start cytoreductive therapy, exhibit a more pronounced

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chronic proinflammatory state than others, which could imply inadequate absorption of

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vitD3 and a decrease in the levels of 25(OH)D transporter proteins (Buison et al.32). Some authors state that inadequate levels of 25(OH)D could be related to a chronic

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inflammatory condition that would also cause lower levels of HDL cholesterol (HDL-

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C). It was demonstrated that low levels of HDL-C could constitute a prognostic marker for hemolysis and endothelial dysfunction given its anti-inflammatory, antioxidant,

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antiaggregant, anticoagulant and profibrinolytic properties33. Mandese et al.30 asserted that the severity of the disease could influence the anthropometric abnormalities of these patients, and Al-Saqladi et al.34 reported that almost two-thirds of patients with SCD exhibit below normal values for some growth parameters, such as height, weight and body mass index (BMI). A study of endocrine/metabolic complications described an incidence of 92%, mainly as a result of insufficient or deficient levels of 25(OH)D30. Although age > 10 years and the dosage of 50,000 IU/month showed a trend towards statistical significance as a risk factor for not reaching optimal levels of 25(OH)D, with ORs of 3.66 and 4.00, respectively, the multivariate analysis revealed that the 800 IU/day regimen provided the most "protection" to reach sufficient levels. To the best of

Journal Pre-proof our knowledge, this is the first study to provide supportive evidence that daily vitD supplementation helps SCD patients achieve adequate levels of 25(OH)D, although no differences were observed in the rest of the studied parameters, including BMD and its annual gain or loss. The American Academy of Pediatrics recently included SCD in the list of hematological diseases at risk for secondary osteoporosis20. Current guidelines recommend maintaining 25(OH)D levels > 20 ng/ml to prevent the risk of bone

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fractures in children35. Therefore, our results are encouraging.

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This study has some important limitations. It is a single-center study with a limited

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sample size. Not all patients had two DXA studies. An optimal level of 25(OH)D ≥ 30

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ng/ml was considered, as in most publications on SCD, although recently the global consensus on the prevention and management of nutritional rickets defined sufficiency

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as levels > 20 ng/ml22. The patients did not begin the prophylaxis program at the same

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age (instead, they began it when the program was approved for them); consequently, there are important age differences, and we know that some of our variables, such as

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BMD, are strongly influenced by age. Therefore, a longitudinal study in which 25(OH)D levels are measured at the same rate and the DXA evaluations have a predetermined schedule could provide more information on the evolution of the examined parameters. Currently, there are no clinical trials in this population that evaluate the benefits of vitD3 supplementation versus placebo, so the evidence is still insufficient. Studies such as this one are essential to continue advancing in the knowledge of this population and to improve their quality of life. In conclusion, vitD3 prophylaxis is a safe practice in patients with SCD and improves the levels of 25(OH)D in this population. As dictated by the universal recommendations, in our population area, a prophylaxis begins with 400 IU/day from

Journal Pre-proof the first month of life and until the infant turns one year old. With the results of our study, we recommend, after completing this period, continue with a 800 IU dose daily of vitD3 in younger children, stopping during summer months. Although daily regimen with 800 IU could be more effective for reaching levels ≥ 30 ng/ml, we tend to think than a 25 000 UI monthly dose will favor adherence to treatment, especially preadolescent and adolescent patients. It is especially in this age range that we should raise awareness about the importance of good bone health. In addition, the severity of

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the evolution of the disease can influence the achievement of optimal 25(OH)D levels.

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Clinical trials to evaluate the effectiveness of vitD3 supplementation and changes in

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bone health after the start of supplementation versus placebo are crucial for patients

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with SCD.

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COLLABORATORS

Cristina Beléndez M.D.

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Collaborators of the VIT-SICKLE-Study Group:

Madrid, Spain

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Pediatric Hematology Unit, “Hospital General Universitario Gregorio Marañón”

Cristina Mata-Fernández M.D., Ph.D. Pediatric Hematology Unit, “Hospital General Universitario Gregorio Marañón” Madrid, Spain

Jorge Huerta-Aragonés M.D. Pediatric Hematology Unit, “Hospital General Universitario Gregorio Marañón” Madrid, Spain

Laura Escobar-Fernández M.D. Pediatric Hematology Unit, “Hospital General Universitario Gregorio Marañón” Madrid, Spain

Journal Pre-proof Cristina Béliz-Mendiola N.P. Pediatric Hematology Unit, “Hospital General Universitario Gregorio Marañón”

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Madrid, Spain

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REFERENCES

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1. Holick MF. Vitamin D deficiency. N Engl J Med. 2007;357(3):266-81. 2. Giustina A, Adler RA, Binkley N, Bouillon R, Ebeling PR, Lazaretti-Castro M, et al.

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Controversies in Vitamin D: Summary Statement From an International Conference. J

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Clin Endocrinol Metab. 2019;104(2):234-240. 3. Goulding A, Jones IE, Taylor RW, Williams SM, Manning PJ. Bone mineral density

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and body composition in boys with distal forearm fractures: a dual-energy x-ray absorptiometry study. J Pediatr. 2001;139(4):509-15. 4. Winzenberg T, Powell S, Shaw KA, Jones G. Effects of vitamin D supplementation on bone density in healthy children: systematic review and meta-analysis. BMJ. 2011;342:c7254. 5. Lobitz S, Telfer P, Cela E, Allaf B, Angastiniotis M, Backman Johansson C, et al; with the endorsement of EuroBloodNet, the European Reference Network in Rare Haematological Diseases. Newborn screening for sickle cell disease in Europe: recommendations from a Pan-European Consensus Conference. Br J Haematol. 2018;183(4):648-660.

Journal Pre-proof 6. Piel FB, Steinberg MH, Rees DC. Sickle Cell Disease. N Engl J Med. 2017;376(16):1561-1573. 7. Ganguly A, Boswell W, Aniq H. Musculoskeletal manifestations of sickle cell anaemia: a pictorial review. Anemia. 2011;2011:794283. 8. Rovner AJ, Stallings VA, Kawchak DA, Schall JI, Ohene-Frempong K, Zemel BS. High risk of vitamin D deficiency in children with sickle cell disease. J Am Diet Assoc. 2008;108(9):1512-6.

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9. Bell NH, Greene A, Epstein S, Oexmann MJ, Shaw S, Shary J. Evidence for

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alteration of the vitamin D-endocrine system in blacks. J Clin Invest. 1985;76(2):470-3.

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10. O'Connor MY, Thoreson CK, Ramsey NL, Ricks M, Sumner AE. The uncertain

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significance of low vitamin D levels in African descent populations: a review of the bone and cardiometabolic literature. Prog Cardiovasc Dis. 2013;56(3):261-9.

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11. Garrido C, Cela E, Beléndez C, Mata C, Huerta J. Status of vitamin D in children

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with sickle cell disease living in Madrid, Spain. Eur J Pediatr. 2012;171(12):1793-8. 12. Nolan VG, Nottage KA, Cole EW, Hankins JS, Gurney JG. Prevalence of vitamin D

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deficiency in sickle cell disease: a systematic review. PLoS One. 2015;10(3):e0119908. 13. Lee MT, Licursi M, McMahon DJ. Vitamin D deficiency and acute vaso-occlusive complications in children with sickle cell disease. Pediatr Blood Cancer. 2015;62(4):643-7.

14. Osunkwo I, Hodgman EI, Cherry K, Dampier C, Eckman J, Ziegler TR, et al. Vitamin D deficiency and chronic pain in sickle cell disease. Br J Haematol. 2011;153(4):538-40. 15. Chapelon E, Garabedian M, Brousse V, Souberbielle JC, Bresson JL, de Montalembert M. Osteopenia and vitamin D deficiency in children with sickle cell disease. Eur J Haematol. 2009;83(6):572-8.

Journal Pre-proof 16. Wang TJ, Pencina MJ, Booth SL, Jacques PF, Ingelsson E, Lanier K, et al. Vitamin D deficiency and risk of cardiovascular disease. Circulation. 2008;117(4):503-11. 17. Gupta A, Sjoukes A, Richards D, Banya W, Hawrylowicz C, Bush A, et al. Relationship between serum vitamin D, disease severity, and airway remodeling in children with asthma. Am J Respir Crit Care Med. 2011;184(12):1342-9. 18. Levin A, Bakris GL, Molitch M, Smulders M, Tian J, Williams LA, et al. Prevalence of abnormal serum vitamin D, PTH, calcium, and phosphorus in patients

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Kidney Int. 2007;71(1):31-8.

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19. Kalkwarf H.J, Abrams S.A, Di MeglioL. A, Koo W.K, Specker B.L, Weiler H.

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Bone Densitometry in Infants and Young Children: The 2013 ISCD Pediatric Official Positions. J of Clin Den 2014; 17(2):243-257

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20. Bachrach LK, Gordon CM, AAP SECTION ON ENDOCRINOLOGY. Bone

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Densitometry in Children and Adolescents. Pediatrics. 2016;138(4):e20162398. 21. Cela E, Ruiz A, Cervera A, et al. Guía de Práctica Clínica de la Enfermedad de

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Células Falciformes. Sociedad Española de Hematología y Oncología Pediátrica (SEHOP). Madrid: Ediciones CeGe. 2019. ISBN 978-84-944935-5-3. 22. Munns CF, Shaw N, Kiely M, Specker BL, Thacher TD, Ozono K, et al. Global Consensus Recommendations on Prevention and Management of Nutritional Rickets. J Clin Endocrinol Metab. 2016;101(2):394-415. 23. Forrest KY, Stuhldreher WL. Prevalence and correlates of vitamin D deficiency in US adults. Nutr Res. 2011;31(1):48-54. 24. Shipton EE, Shipton EA. Vitamin D Deficiency and Pain: Clinical Evidence of Low Levels of Vitamin D and Supplementation in Chronic Pain States. Pain Ther. 2015;4(1):67-87.

Journal Pre-proof 25. Han J, Zhang X, Saraf SL, Gowhari M, Molokie RE, Hassan J, et al. Risk factors for vitamin D deficiency in sickle cell disease. Br J Haematol. 2018;181(6):828-835. 26. Arlet JB, Courbebaisse M, Chatellier G, Eladari D, Souberbielle JC, Friedlander G, et al. Relationship between vitamin D deficiency and bone fragility in sickle cell disease: a cohort study of 56 adults. Bone. 2013;52(1):206-11. 27. Sadat-Ali M, Al-Elq A, Al-Turki H, Sultan O, Al-Ali A, AlMulhim F. Vitamin D level among patients with sickle cell anemia and its influence on bone mass. Am J

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28. Barden EM, Kawchak DA, Ohene-Frempong K, Stallings VA, Zemel BS. Body

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composition in children with sickle cell disease. Am J Clin Nutr. 2002;76(1):218-25.

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29. Singhal A, Parker S, Linsell L, Serjeant G. Energy intake and resting metabolic rate

2002;75(6):1093-7.

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in preschool Jamaican children with homozygous sickle cell disease. Am J Clin Nutr.

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30. Mandese V, Bigi E, Bruzzi P, Palazzi G, Predieri B, Lucaccioni L, et al. Endocrine and metabolic complications in children and adolescents with Sickle Cell Disease: an

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Italian cohort study. BMC Pediatr. 2019;19(1):56. 31. Garrido Colino C, Beléndez Bieler C, Pérez Díaz M, Cela de Julián E. [Evaluation of bone mineral density in children with sickle cell disease]. An Pediatr (Barc). 2015;82(4):216-21.

32. Buison AM, Kawchak DA, Schall J, Ohene-Frempong K, Stallings VA, Zemel BS. Low vitamin D status in children with sickle cell disease. J Pediatr. 2004;145(5):622-7. 33. Seixas MO, Rocha LC, Carvalho MB, Menezes JF, Lyra IM, Nascimento VM, et al. Levels of high-density lipoprotein cholesterol (HDL-C) among children with steadystate sickle cell disease. Lipids Health Dis. 2010;9:91.

Journal Pre-proof 34. Al-Saqladi AW, Cipolotti R, Fijnvandraat K, Brabin BJ. Growth and nutritional status of children with homozygous sickle cell disease. Ann Trop Paediatr. 2008;28(3):165-89. 35. Ward L.M, Konji V.N, Ma J. The management of osteoporosis in children.

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Osteoporos Int 2016;27:2147-2179.

Journal Pre-proof Contributors’ Statements: Carmen Garrido  CRediT roles: Conceptualization; investigation; Methodology; Project administration; Supervision; Validation. Roles: Writing – review and editing. Eduardo J. Bardón-Cancho  CRediT roles: Data curation; Formal analysis; Investigation; Methodology; Project administration; Software; Visualization. Roles: Writing – original draft. Verónica de los Ángeles Fajardo-Sánchez  CRediT roles: Data curation; Formal

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analysis; Investigation. Roles: Writing – original draft.

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María Elena Cascón Pérez-Teijón  CRediT roles: Investigation; Methodology; Project

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administration; Resources; Visualization. Roles: Writing – review and editing.

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Marina García-Morin  CRediT roles: Data curation; Resources; Validation. Roles:

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Writing – review and editing.

Elena Cela  CRediT roles: Project administration; Supervision; Validation. Roles:

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Writing – review and editing.

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VIT-SICKLE Study Group  CRediT roles: data curation; Resources.

All authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work.

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p25-p75 7.44-14.59 1.04-7.98 48.28-71.00 21.57-37.75 23.95-36.75 25.40-38.40 26.67-38.40 6.07-34.00

64 51 64 52 52 52 52

-1.10 – 0.60 -1.30 - 0.1 0.51-0.77 0.45-0.64 13-57 3.40-16.62 1.18-7.28

-0.35 ± 1.22 -0.55 ± 1.10 0.62 ± 0.16 0.55 ± 0.13 32.79 ± 24.02 9.62 ± 9.12 4.51 ± 4.43

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of N 80 80 80 72 77 79 80 80

n (%) 36 (45.0) 44 (55.0) 53 (66.3) 26 (32.5) 1 (1.3) 39 (48.8) 19 (23.8) 15 (18.8) 70 (87.5) 5 (6.3) 2 (2.5) 3 (3.8) 62 (77.5) 15 (18.8) 3 (3.8) 52 (67.5) 10 (13.0) 15 (19.5) 66 (82.5) 64 (80.0) 48 (94.2) 44 (55.0) 20 (25.0) M ± SD 11.06 ± 4.41 5.06 ± 4.35 59.05 ± 16.88 29.84 ± 11.80 30.29 ± 10.76 32.21 ± 9.73 32.57 ± 9.20 21.39 ± 18.36

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Sex: female male Race: African African American Hindu Treatment with hydroxyurea Transfusion regimen HPCT Type of hemoglobinopathy: SS SC Sβ+ Sβ0 VitD3 prophylaxis frequency: Monthly Daily Not recorded Monthly dose: 25,000 IU 50,000 IU Daily dose: 800 IU Optimal levels (≥ 30) reached At least 1 DXA Maintenance or gain in BMD Z-Score ≥ 10 years of age Prophylactic vitD3 started in infancy

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Current age (years) Age at start of prophylactic vitD3 (years) VitD3 prophylaxis (months) 25(OH)D 1 25(OH)D 2 25(OH)D 3 25(OH)D 4 Time required to reach optimal levels (≥ 30) (months) BMD Z-score 1 BMD Z-score 2 BMD 1 (g/cm2) BMD 2 (g/cm2) Time between DXA measurements (months) BMD gain/loss (%) Annual BMD gain/loss (%)

Variance 19.43 18.91 284.85 139.20 115.83 94.76 84.67 337.07 1.50 1.21 0.02 0.02 577.11 83.20 19.67

TABLE 1. Summary of epidemiological, analytical, BMD Z-score and bone mineral density (BMD) data for children with sickle cell disease (SCD) (n = 80). 25(OH)D 1 is the oldest measurement, and 25(OH)D 4 is the most recent; BMD Zscore 1 and BMD 1 correspond to the 1st densitometry measurement performed

Journal Pre-proof after the start of vitD3 prophylaxis, and BMD Z-score 2 and BMD 2 correspond to the 2nd or most recent densitometry measurement. HPCT: hematopoietic progenitor cell transplant; VitD3: vitamin D; IU: international units; DXA: bone densitometry; 25(OH)D: 25-hydroxyvitamin D; M

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± SD: mean ± standard deviation; BMD: bone mineral density.

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25(OH)D 1 25(OH)D 2 25(OH)D 3 25(OH)D 4

Start of prophylaxis in infancy Yes No Yes No Yes No Yes No

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Mean (ng/ml)

54 18 58 19 59 20 60 20

35.71 27.89 34.57 28.89 38.20 30.18 36.35 31.31

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0.014 0.045 0.001 0.033

TABLE 2. Analysis of the differences between 25(OH)D levels as a function of age

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at the onset of vitD3 prophylaxis, where 25(OH)D 1 is the oldest measurement, and

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25(OH)D 4 is the most recent.

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25(OH)D: 25-hydroxyvitamin D; vitD3: vitamin D3

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Univariate analysis

OR (95% CI)

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OR (95% CI)

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14 (21.1)

1 (7.1)

0.28 (0.03-2.37)

0.246

0.03 (0.00-0.91)

0.044

45 (68.2) 6 (9.1)

7 (50.0) 4 (28.6)

0.47 (0.14-1.49) 4.00 (0.96-16.67)

0.201 0.058

0.12 (0.01-1.67) 0.29 (0.02-4.63)

0.114 0.385

20 (30.3)

0 (0.0)

NA

NA

45 (68.2) 20 (30.3) 1 (1.5) 36 (54.5)

8 (57.1) 6 (42.9) 0 (0.0) 8 (57.1)

0.62 (0.19-2.02) 1.72 (0.53-5.62) NA 0.90 (0.28-2.88)

0.430 0.366 NA 0.859

0.54 (0.14-2.08)

0.376

30 (45.5)

9 (64.3)

15 (22.7)

4 (28.6)

0.73 (0.20-2.68)

0.642

15 (22.7) 60 (90.9) 33 (50.0)

0 (0.0) 13 (92.9) 11 (78.6)

NA 0.77 (0.08-6.94) 3.66 (0.94-14.28)

NA 0.815 0.062

3.00 (0.59-15.38)

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Did not reach optimal levels N (%)

2.16 (0.65-7.14)

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Prophylaxis regimen 800 IU/day (12 drops) 25,000 IU/month 50,000 IU/month Started prophylaxis in infancy Race African African American Hindu Male sex Treatment Hydroxyurea Transfusion regimen HPCT Hb SS + Sβ0 Age > 10 years

Reached optimal levels N (%)

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Variable

Multivariate analysis

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TABLE 3. Risk factors for not reaching optimal 25(OH)D levels. Univariate and multivariate analyses. The data are expressed as absolute frequency and percentage

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analysis are in bold.

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with respect to reaching optimal levels. The variables included in the multivariate

IU: international units; OR: odds ratio; CI: confidence interval; NA: not assessable due to observed frequency = 0

FIGURE 1. Frequency (N and %) according to the categorization of 25(OH)D levels (regardless of dose and prophylaxis regimen used), where level 1 corresponds to the oldest measurement, and level 4 corresponds to the most recent. <10 ng/ml: severe deficit; 10-19.9 ng/ml: moderate deficit; 20-29.9 ng/ml: insufficiency; ≥ 30 ng/ml: optimal levels. 0 FIGURE 2. Time curves until reaching optimal 25(OH)D levels (Kaplan-Meier). A. Curve in function of age older than or younger than 10 years; B. Curve in function of treatment with hydroxyurea.

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Journal Pre-proof HIGHLIGHTS



In Sickle Cell Disease there is a greater probability of vitamin D deficiency than general population.



Prophylaxis with vitamin D3 is a safe practice in patients with sickle cell disease.



It is especially important to start this prophylaxis in breastfeeding period and keep it beyond the first year of life.

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Daily schedule with 800 IU could be more effective for reaching vitamin D levels ≥ 30 ng/ml.

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In pre-adolescent and adolescent patients is especially where we should raise awareness

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of the care of a damaged bone health.

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