The association between fat mass and obesity‐associated (FTO) genotype and serum vitamin D level in breast cancer patients

Abstract The preventive effect of vitamin D against breast cancer can be influenced by gene polymorphisms. This study aimed to investigate the association between serum level of 25(OH) vitamin D and FTO genotype in breast cancer patients. A cross‐sectional study was carried out on 180 newly diagnosed patients with breast cancer in Tehran, Iran. The blood samples were collected from the participants in order to assess the FTO gene rs9939609 polymorphism by the tetra‐primer amplification refractory mutation system (Tetra‐ARMS) PCR method. The serum level of 25(OH) vitamin D was measured using the direct competitive enzyme‐linked immunosorbent assay (ELISA) method. The association between vitamin D and the FTO genotype in patients with breast cancer was assessed after adjustment for cofounders. The frequency of TT, AT and AA genotypes in the breast cancer patients were 43% (n = 77), 49% (n = 89) and 8% (n = 14), respectively. All patients with higher than 40 ng/dl of serum 25(OH) vitamin D had one or two copies of FTO rs9939609 risk allele (p = 0.019). No linear association was found between the number of FTO risk allele and the level of serum vitamin D. All patients with high serum level of 25(OH) vitamin D had one or two copies of FTO rs9939609 risk allele. FTO gene polymorphisms may counteract the beneficial effects of vitamin D in breast cancer prevention. Further studies can help to better understand the genetic factors predisposing to breast cancer and their effect on the association between vitamin D and breast cancer.


| Study population and data collection
This cross-sectional study included 180 newly diagnosed patients with BC and performed from January 2019 to August 2020. The sample size was calculated using Open EPI software and similar previous studies. 16,17 The participants were selected according to the inclusion criteria from the female patients referring to the cancer research centre of Shohadaye Tajrish Hospital in Tehran, Iran. The inclusion criteria included females with confirmed BC, diagnosed recently, age between 35 and 70 years and consent to participate in the study. Basic information including medical history, alcohol consumption, smoking, the level of education and socioeconomic factors was collected. The amount of calorie and vitamin D intake in the past year was assessed using a validated Food Frequency Questionnaire (FFQ). 18 The level of physical activity was measured using the International Physical Activity Questionnaire (IPAQ) which was validated in a previous study. 19 The patient's weight and height were measured using a SECA scale (Alpha 882, SECA Corporation, Hamburg, Germany), and their height was measured using a stadiometer, respectively. The BMI was calculated by dividing the weight (kg) by the square of height (m 2 ).

| Genotyping
At the beginning of the study, from each participant, about 5 ml intravenous blood sample was collected onto an ethylenediaminetetraacetic acid (EDTA) tube (Becton Dickinson, France) by a trained phlebotomist. The blood sample was stored at −70 C until used for further analysis. The genomic deoxyribonucleic acid (DNA) was extracted from the blood samples using GeneAll DNA extraction kit (Incheon, Korea) following the manufacturer's instruction. Polymerase chain reaction (PCR) was performed with 50 ng of genomic DNA using a PCR amplification instrument (GeneQ; Hangzhou Bioer Technology Co., Ltd.) and master mix DNA polymerase (cat. No A180301; Ampliqon). The PCR products were examined to identify rs9939609 polymorphism of the FTO gene by the tetra-primer amplification refractory mutation system (Tetra-ARMS) PCR method. vitamin D. All patients with high serum level of 25(OH) vitamin D had one or two copies of FTO rs9939609 risk allele. FTO gene polymorphisms may counteract the beneficial effects of vitamin D in breast cancer prevention. Further studies can help to better understand the genetic factors predisposing to breast cancer and their effect on the association between vitamin D and breast cancer.

| RE SULTS
The frequency of TT, AT and AA genotypes were 43% (n = 77), 49%  Table 1. The mean age of TT carriers and AT/AA carriers were 59.57 (±11.66) and 57.73 (±11.11), respectively (p > 0.05). No significant difference was found in terms of the anthropometry (height, weight and BMI), breastfeeding, menopause age, sleep duration stage of cancer, pregnancy, marital status, family history of BC, menopause and number of abortions between the patients with TT and AA/AT genotypes. However, when serum vitamin D levels were assessed, significant differences were evident when the patients were characterized based on the pregnancy (p < 0.05) and abortion history (p < 0.01) ( Table 1).
The distribution of the status of lifestyle and dietary intake among different FTO genotypes and different serum levels of vitamin D are presented in Table 2. No significant difference was evident on smoking, alcohol consumption sleep duration, macronutrient/energy intake or blood serum level of vitamin D between TT and AA/ AT genotypes. Based on the assessment of serum vitamin D levels and macronutrient intake characteristics, it was evident that the participants with a low level of vitamin D had a significantly (p < 0.01) higher daily intake of carbohydrates. All patients with higher than 40 ng/dl of serum 25(OH) vitamin D had one or two copies of FTO rs9939609 risk allele (p = 0.019) ( Table 2).
No linear association was identified between the number of FTO risk allele and the level of serum vitamin D (