www.nature.com/scientificreports OPEN Association of BET1L and TNRC6B with uterine leiomyoma risk and its relevant clinical features in Han Received: 30 January 2018 Accepted: 27 April 2018 Chinese population Published: xx xx xxxx Bailing Liu1,2, Tao Wang3, Jue Jiang1, Miao Li1, Wenqi Ma1, Haibin Wu3 & Qi Zhou1 Previous studies have shown that uterine leiomyomas (UL) are benign tumours with contributions from environmental and genetic factors. We aimed to replicate two initial signifcant genetic factors, TNRC6B and BET1L, in a Han Chinese population. A total of 2,055 study subjects were recruited, and 55 SNPs mapped to TNRC6B and BET1L were selected and genotyped in samples from these subjects. Genetic associations were analysed at both the single marker and haplotype levels. Associations between targeted SNPs and relevant clinical features of UL were analysed in case only samples. Functional consequences of signifcant SNPs were analysed by bioinformatics tools. Two SNPs, rs2280543 from BET1L (χ2 = 18.3, OR = 0.64, P = 1.87 × 10−5) and rs12484776 from TNRC6B (χ2 = 19.7, OR = 1.40, P = 8.91 × 10−6), were identifed as signifcantly associated with the disease status of UL. Rs2280543 was signifcantly associated with the number of fbroid nodes (P = 0.0007), while rs12484776 was signifcantly associated with node size (χ2 = 54.88, P = 3.44 × 10−11). Both SNPs were a signifcant eQTL for their genes. In this study, we have shown that both BET1L and TNRC6B contributed to the risk of UL in Chinese women. Signifcant SNPs from BET1L and TNRC6B were also identifed as signifcantly associated with the number of fbroid nodes and the size of the node, respectively. Uterine leiomyomas (UL), also known as uterine fbroids, are benign tumours in the uterus1. UL are typically identifed during the mid or late reproductive years in women, and they decrease in size afer menopause2. Te size and number of fbroids can vary among patients3. According to a systematic review published in 2017, the incidence of UL could be ranged from 217–3745 cases per 100,000 women-years4. In general, the prevalence of UL in women ranges from 20% in Europeans to as high as 80% in African-American women4,5. No symptoms can be identifed in more than 50% of women with UL6. For the remaining women with UL, their clinical symptoms can range from abnormal bleeding and pelvic pain to infertility and pregnancy complications6. Evidence from multiple studies has shown that UL have contributions from both environmental and genetic factors7–9. Early familial aggregation and twin studies have identifed a signifcant genetic component to UL pre- disposition10,11. Makinen et al. performed a whole-exome sequencing study on 18 UL patients and identifed the MED12 gene as contributing to tumourigenesis12. In a GWAS based on Japanese populations conducted in 2011, three loci on chromosomes 10q24.33, 22q13.1, and 11p15.5 were identifed to be signifcantly associ- ated with the disease status of UL13. Tese three loci included several genes such as STE20 Like Kinase (SLK), Oligosaccharide-Binding Fold-Containing Protein 1 (OBFC1), Trinucleotide Repeat Containing 6B (TNRC6B), Outer Dense Fiber 3 (ODF3), Bet1 Golgi Vesicular Membrane Trafcking Protein Like (BET1L), RIC8 Guanine Nucleotide Exchange Factor A (RIC8A), and Sirtuin 3 (SIRT3). Since then, several follow up studies have tried to replicate these initial GWAS fndings using study samples based on other ethnic groups14–16. However, the results of these subsequent studies have not been concordant and have, at times, been contradictory. More studies with large sample sizes are still needed to confrm these previous hits. In this study, we attempted to replicate two initial signifcant loci, TNRC6B and BET1L, identifed in a GWAS conducted by Cha et al.13 by using study subjects with Chinese Han ancestry. A total of 2,055 study subjects were 1Department of Ultrasound, the Second Affiliated Hospital, Xi’an Jiaotong University, Xi’an, Shaanxi, China. 2Department of Ultrasound, Children’s Hospital of Xi’an, Xi’an, Shaanxi, China. 3Department of Pediatrics, Children’s Hospital of Xi’an, Xi’an, Shaanxi, China. Correspondence and requests for materials should be addressed to Q.Z. (email: [email protected]) SCIENTIFIC REPORTS | (2018) 8:7401 | DOI:10.1038/s41598-018-25792-z 1 www.nature.com/scientificreports/ Subjects (N = 2,055) Characteristics UL patients (N = 674) Controls(N = 1,381) P-value Age, mean ± SD (years) 38.3 ± 7.22 38.2 ± 7.34 0.78 BMI, mean ± SD (kg/m2) 23.7 ± 1.37 23.6 ± 1.30 0.12 Married (%) 647 (96.0) 1328 (96.2) 0.94 Age of menarche, mean ± SD (years) 13.5 ± 1.47 13.4 ± 1.38 0.12 Duration of menses, mean ± SD (days) 6.5 ± 1.52 6.7 ± 1.69 0.005 Menstrual cycle, mean ± SD (days) 28.8 ± 1.39 28.6 ± 1.67 0.003 Bleeding (%) 396 (58.5) NA NA Pain (%) 225 (33.4) NA NA Fibroid node (%) NA NA Single 228(33.8) Multiple 446(66.2) Node size (%) NA NA Small 79(11.7) Medium 434(64.4) Large 161(23.9) Table 1. Te clinical and demographic characteristics of the uterine leiomyoma and control groups. UL: uterine leiomyoma; BMI: body mass index; SD: standard deviation; NA: not available. recruited, and 55 SNPs mapped to TNRC6B and BET1L were selected and genotyped in samples from these subjects. In addition to genetic associations between these SNPs and the disease status of UL, we also examined potential associations between targeted SNPs and clinical characteristics of UL. Bioinformatics tools were also utilized to evaluate the potential biological functions of the targeted SNPs. Methods Study subjects. In the present study, a total of 674 women with UL and 1,381 healthy women, controls without any systematic disease, were recruited from the Second Afliated Hospital of Xi’an Jiaotong University between April 2013 and May 2017. All patients were diagnosed with UL by ultrasonography and confrmed by at least two senior physicians, and all subjects were screened for no other female reproductive system tumours, systemic disease or history of malignancy. Self-administered questionnaires were used to collect demographic data, and the characteristics of our study subjects are shown in Table 1. All participants were unrelated Han Chinese individuals, and the UL and control groups were matched by age and body mass index (BMI). Signifcant diferences were identifed for duration of menses (P = 0.005) and menstrual cycle (P = 0.003) between UL cases and healthy controls. Te size of UL was categorized into three groups (small, medium, and large) based on the diameter of the UL (small ≤2 cm, 2 cm <medium <4 cm, large ≥4 cm). If subjects were diagnosed with multiple UL, the largest one determined the size group. Te study protocol was approved by the Ethics Committee of Xi’an Jiaotong University in accordance with the ethical guidelines of the Declaration of Helsinki of 1975 (revised in 2008). Written informed consent was obtained from participants. SNP selection and Genotyping. We searched for all SNPs with a minor allele frequency (MAF) ≥0.05 within the regions of the TNRC6B and BET1L genes in the 1000 Genomes Chinese Han Beijing population (CHB). Ten, MAF ≥0.05 with pair-wise tagging and r2 ≥0.8 were used as the cut-of criteria during tag SNP selection, which generated 27 and 28 tag SNPs within the TNRC6B and BET1L genes, respectively. General infor- mation about these 55 selected SNPs is summarized in Supplemental Table S1. Most of the selected SNPs were non-coding SNPs. Genomic DNA was extracted from peripheral blood leukocytes according to the manufac- turer’s protocol (Genomic DNA kit, Axygen Scientifc Inc., California, USA). Genotyping was performed for all SNPs using the Sequenom Mass ARRAY RS1000 system (Sequenom, San Diego, California, USA). Te results were processed using Typer Analyser sofware, and genotype data were generated from the samples17. Case and control status was blinded during all genotyping processes for quality control. Five percent of the samples were repeated at random, and the results were 100% concordant. Statistical and Bioinformatics Methods. Hardy-Weinberg equilibrium was tested for each SNP within the control samples. χ2 tests were performed for each SNP to evaluate the diferences in allelic and genotypic distributions between UL cases and controls. Linkage disequilibrium (LD) blocks were constructed for both genes, and haplotype-based analyses were conducted for each block. Plink was utilized for the analyses mentioned above18. In addition to genetic association analyses focusing on disease status, we also analysed the potential link between signifcant SNPs and four clinical features of UL, including bleeding, pain, number of fbroid nodes, and size of the node, in a subset of our samples that included UL cases only. χ2 tests were performed for these analyses. In general, Bonferroni correction was applied to address multiple comparisons. For single marker-based association analyses, the threshold P value was 0.05/55 ≈ 9 × 10−4. Genomic control was applied to correct for the potential efects of population stratifcation19,20. Te null distribution of genomic infation factor λ was con- structed by 10,000 bootstrapping. SCIENTIFIC REPORTS | (2018) 8:7401 | DOI:10.1038/s41598-018-25792-z 2 www.nature.com/scientificreports/ Allelic Test Genotypic Test rs2280543(%) T (N = 538) C (N = 3,572) OR χ2 P* TT (N = 37) CT (N = 464) CC (N = 1,554) χ2** P* Patients 133 (9.8) 1,215 (90.2) 3 (0.4) 127 (18.8) 544 (80.8) Controls 405 (14.6) 2,357 (85.4) 0.64 18.3 1.87 × 10−5 34 (2.5) 337 (24.4) 1,010 (73.1) — 1.85 × 10−5 rs12484776(%) G (N = 999) A (N = 3,111) GG (N = 125) GA (N = 749) AA (N = 1,181) Patients 385 (28.6) 963 (71.4) 61 (9.1) 263 (39.0) 350 (51.9) Controls 614 (22.2) 2,148 (77.8) 1.40 19.7 8.91 × 10−6 64 (4.6) 486 (35.2) 831 (60.2) 21.7 1.94 × 10−5 Table 2.