We will conduct a systematic review and meta-analysis following the principles of the Cochrane Handbook for Systematic Reviews of Intervention  and report the data following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement  or the Preferred Reporting Items for Systematic Reviews and Meta-Analyses for Children (PRISMA-C) which will be published soon. This protocol follows the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocol 2015 (PRISMA-P), which is included as Additional file 1 .
The patient, intervention, comparator, outcome (PICO) question formulated for this study will be as follows: In children aged less than 18 years of age (P), do the CYP2C9, VKORC1, and CYP4F2 genetic polymorphism (I) compared with the CYP2C9, VKORC1, and CYP4F2 genetic wild type (C) impact on the variability of maintenance dose of patient receiving warfarin (O)?
The detail of the outcome is as follows:
Mean difference of warfarin maintenance dose between CYP2C9 variant type (CYP2C9 *1/*2, CYP2C9 *1/*3, CYP2C9 *2/*2, CYP2C9 *2/*3, or CYP2C9 *3/*3) and wild type (CYP2C9 *1/*1) in children
Mean difference of warfarin maintenance dose between VKORC1 variant type (VKORC1-1639GA, VKORC-1639AA, VKORC1-1173CT, or VKORC1-1173CT) and VKORC wild type (VKORC1-1639GG or VKORC1-1173TT) in children
Mean difference of warfarin maintenance dose between CYP4F2 variant type (CYP4F2-CT or CYP4F2-TT) to that in the CYP4F2 wild type (CYP4F2-CC) in children
We will combine the data regarding haplotypes of VKORC1-1173C>T and VKORC1-1639G>A because these two SNPs have a strong linkage disequilibrium . The unit of mean difference of warfarin maintenance dose will be expressed as milligrams per kilogram per day. When warfarin maintenance dose, which is standardized to milligrams per kilogram per day, is not applicable, we will use standardized mean differences with 95 % confidence interval (CI) for synthesis. Also, we will conduct subgroup analysis according to the literature using milligrams per kilogram per day as the outcome measure.
Search strategy and sources
A comprehensive literature review search using the OVID platform will be conducted by a specialized librarian, including MEDLINE, EMBASE, Cochrane Central Register of Controlled Trial libraries, as well as gray literature. We will identify relevant studies in any language using medical subject headings (MeSH terms) and text words related to “warfarin” AND “target SNPs” AND “children.” Details of the search strategy are provided in Additional file 2. An age filter (all children, 0 to 18 years) will be used in the MEDLINE and EMBASE plus EMBASE classics search. We will also include studies published only as abstract or conference reports. Additionally, we will hand-search the reference lists of each eligible study included in the present and previous systematic reviews to identify additional eligible articles. In addition, the International Clinical Trials Registry Platform Search Portal and ClinicalTrials.gov will be searched for ongoing or recently completed trials and the international prospective register of systematic reviews (PROSPERO) will be searched for ongoing or recently completed systematic reviews. As relevant studies are identified, reviewers will check for additional relevant cited and citing articles. The principal investigators of studies will be contacted to clarify study eligibility if required. Primary authors will also be contacted to ascertain missing or unpublished data and their knowledge of any other studies not retrieved by our primary search.
Our systematic review protocol was registered with the PROSPERO on 27 February 2015 (registration number: CRD42015016172). If this protocol needs any amendments, we will provide the date of each amendment, describe the change, and give the rationale, accordingly. Changes will not be incorporated into the protocol.
This review will focus on the potential association between warfarin maintenance dose and genotypes of CYP2C9, VKORC1, or CYP4F2 in children aged less than 18 years old. We will define warfarin maintenance dose as the dose to achieve three consecutive international normalized ratio (INR) measurements within a therapeutic range over a minimum of 4 weeks. We will include original studies which meet the following criteria: (1) observational (cohort or cross-sectional) studies or randomized controlled trials in warfarin-treated pediatric patients; (2) genotypes of CYP2C9, VKORC1, or CYP4F2 were reported; (3) children receiving warfarin for at least 1 month; and (4) maintenance doses of warfarin are presented according to each genotype. There were no restrictions in the inclusion criteria regarding patient demographic information including body weight, height, use of concurrent drugs, indication for warfarin use, and target INR ranges. We will exclude case–control studies, case reports, case series, reviews, editorials, news, and original studies which did not report the outcome of interest in both the children and adults. Duplicated studies will be also excluded. Pharmacogenetic studies involving only adult population aged 18 or older will be excluded. When a study involves both adult and pediatric patients, we will exclude the study if the mean or median age of the study population exceeded 18 years old and subgroup analysis regarding children is unavailable.
Data collection and analysis
Two reviewers (MT and TK) will independently screen titles and abstracts of all the retrieved bibliographic records. The inclusion and exclusion criteria will be used for each screening step. If no abstract is available, the full text will be obtained unless the article can be confidently excluded by its title alone. If there is any doubt whether a study should be excluded, the study will proceed to the full-text screen to reduce the likelihood of incorrectly excluding relevant studies. Full text of potentially eligible studies will be independently retrieved by the two reviewers. Disagreements at these screening levels (title/abstract and full text) will be resolved through discussion or adjudication of a third reviewer (SI).
After determination of the initial study, eligibility information will be extracted for each study pertaining to study identification (first author, year of publication, and country where patients’ recruitment took place), study design, patient population (number of enrolled patients, age, gender, predominant ethnicity, concomitant drug use, diet, indication for warfarin treatment, target INR range), and primary outcome measurement (allele frequencies and warfarin maintenance dose for each genotype of CYP2C9, VKORC1, or CYP4F2). A list of the eligibility information is presented in Additional file 3. In the case of duplicated studies, we will use the study which had the largest number of patients. We will contact the original authors for missing data if necessary. In order to standardize the unit of warfarin dose to milligrams per kilogram per day, we will also contact the original authors to obtain the data. The two reviewers will independently extract data of all eligible studies and summarize. The disagreement between the reviewers will be resolved by discussion. To ensure consistency across reviewers, we will conduct pre-training of data extraction before starting the review.
Endnote X7 software will be used to upload literature search results and de-duplicated by information regarding author, year of publication, title, and reference type. The Drop Box software, an Internet-based software program that facilitates collaboration among reviewers during the study selection process, will be used to store the literature search results and PDF files of retrieved paper. The uploaded data will be used only for the purposes stated in this study and will not be forwarded to third parties.
Assessing a risk of bias of individual studies
Two reviewers will independently assess the risk of bias of all eligible studies in accordance with the Newcastle–Ottawa Scale (NOS) . In addition, the Strengthening the Reporting of Genetic Association study (STREGA) statement will also be utilized . The STREGA statement represents an extension of the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE)  and advocates for transparency how a study was done by assessing a 12-item checklist which are important to consider in genetic association studies. These will be undertaken by two separate reviewers. Where there is disagreement, a third reviewer will be used as an arbitrator.
Data synthesis and presentation
We will weigh the studies by using the inverse variance method and express the effect of each genetic variant on warfarin maintenance doses as mean differences of the doses between the wild type and the respective variant with 95 % CI. When warfarin maintenance dose, which is standardized to milligrams per kilogram per day, is not applicable, we will use the standardized mean differences with 95 % CI for synthesis. When only the median and range are available after contacting primary authors, an imputation method proposed by Hozo et al.  will be utilized. Anticipating the heterogeneity between studies, we will combine the data using the DerSimonian and Laird random effect model . A two-sided P value less than 0.05 indicates a nominally significant overall association. All statistical analyses will be performed by using the Review Manager version 5.3 (the Nordic Cochrane Centre, Copenhagen, Denmark) or EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan) . If quantitative synthesis is not appropriate, we will provide a systematic narrative synthesis with information presented in the text and tables to summarize and explain the characteristics and findings of the included studies.
We will assess the magnitude of inconsistency in the study results by the I2 statistic which expresses the heterogeneity as the percentage of total variation. We will arbitrarily define the three categories of heterogeneity as follows: I2 <30 %, low; I2 30–70 %, moderate; and I2 >70 %, high. If high levels of heterogeneity among the studies are observed (I2 ≥ 50 %), the study design and characteristics in the included studies will be analyzed. We will try to explain the source of heterogeneity by subgroup analysis or sensitivity analysis.
Publication bias will be assessed using linear regression test of the funnel plot asymmetry. A P value less than 0.10 will be considered to have publication bias. If asymmetry is suggested by a visual assessment, we will perform exploratory analyses to investigate and adjust it (trim and fill analysis).
Planned sensitivity and subgroup analysis
Subgroup analyses will be used to explore possible sources of heterogeneity, based on the following: (1) patient characteristic (age, predominant ethnicity, indication for warfarin); (2) target INR (median target range <2.5 or ≥2.5); and (3) literature using milligrams per kilogram per day as the outcome measure. Sensitivity analyses will be performed in order to explore the source of heterogeneity as follows: (1) quality components (full-text publication vs. abstracts) and (2) quality of studies by omitting studies that are judged to have lower quality.
The proposed review will use the Grading of Recommendations Assessment, Development and Evaluation (GRADE) guidelines to determine the quality and strength of recommendations . Quality will be adjudicated as high (further research is very unlikely to change our confidence in the estimate of effect), moderate (further research is likely to have an important impact on our confidence in the estimate of effect and may change the estimate), low (further research is very likely to have an important impact on our confidence in the estimate of effect and is likely to change the estimate), or very low (very uncertain about the estimate of effect).