Molecular Biology Reports (2019) 46:3299–3306 https://doi.org/10.1007/s11033-019-04790-y

ORIGINAL ARTICLE

Haplotypes of CYP1B1 and CCDC57 in an Afro‑Caribbean female population with uterine leiomyoma

Angela T. Alleyne1 · Virgil S. Bideau1

Received: 13 December 2018 / Accepted: 28 March 2019 / Published online: 13 April 2019 © Springer Nature B.V. 2019

Abstract Uterine leiomyomas (UL) are prevalent benign tumors, especially among women of African ancestry. The disease also has genetic liability and is infuenced by risk factors such as hormones and obesity. This study investigates the haplotypes of the Cytochrome P450 1B1 (CYP1B1) related to hormones and coiled-coil domain containing 57 gene (CCDC57) related to obesity in Afro-Caribbean females. Each haplotype was constructed from unphased sequence data using PHASE v.2.1 software and Haploview v.4.2 was used for linkage disequilibrium (LD) studies. There were contrasting LD observed among the single nucleotide polymorphisms of CYP1B1 and CCDC5. Accordingly, the GTA haplotype of CYP1B1 was signifcantly associated with UL risk (P = 0.02) while there was no association between CCDC57 haplotypes and UL (P = 0.2) for the ATG haplotype. As such, our fndings suggest that the Asp449Asp polymorphism and GTA haplotype of CYP1B1 may contribute to UL susceptibility in women of Afro-Caribbean ancestry in this population.

Keywords SNP · Fibroids · Haplotyping · Estrogen · Linkage disequilibrium

Introduction consistently having a higher risk of UL development than White women [4]. However cumulatively Black women have Globally, 20–25% of women are clinically diagnosed with an earlier onset for diagnosis than White women [2, 10]. uterine leiomyomas (UL); yet some studies suggest that as Genomic analysis of single nucleotide polymorphisms high as 70–80% of women may develop UL within their (SNPs) has been used to identify genetic factors of UL reproductive lifetimes, making these the most commonly susceptibility [12–14]. Despite this, haplotypes of DNA occurring type of tumors in females [1–4]. Although UL are sequence variation defined by multiple SNPs [15] are benign tumors, they pose several risks to women of child- also increasingly being used in studies of human genetics, bearing ages such as abdominal distention, heavy menstrual because they contain more information than individual SNP bleeding and reproductive dysfunction [4–6]. Chromosomal loci [16]. Haplotype association tests take into consideration anomalies support the view that underlying genetic suscepti- the fact that SNPs are not independent of each other; rather, bility factors account for the growth of these non-malignant they tend to be co-inherited and are therefore in linkage dise- neoplasms that afect the female reproductive tract [7–11]. quilibrium (LD) [17]. Haplotypes therefore capture informa- Race and age are two demographic high risk factors for tion about conserved regions in ancestral and developing UL; with women over 40 four times as likely to LD patterns which assist in successful identifcation of rare report the condition than those under 40 and Black women causative variants in single-gene disorders [18, 19]. Notably, African populations or populations with signifcant African Nucleotide sequence data reported are available in the DDBJ/ admixture such as Barbadians [20, 21] have associated hap- EMBL/GenBank databases under the accession number (s) lotype blocks which are narrower, thereby shortening the LT613647-LT613831. list of candidate SNPs to be validated in disease association studies. * Angela T. Alleyne [email protected] Evidence shows that the sex steroid hormone estrogen is an important contributor to tumor growth [22–24]. There- 1 Department of Biological and Chemical Sciences, Faculty fore, allelic variants of genes involved in steroid metabo- of Science and Technology, University of the West Indies lism may infuence a woman’s susceptibility to UL [9, 10, Cave Hill Campus, Bridgetown, Barbados

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23]. Cytochrome P450 1B1 (CYP1B1) being investigated comprised of healthy women who were also being seen by in this study has been haplotyped in two other studies [25, practitioners in the same department. Each participant had 26]. Salimi et al. haplotyped four common SNPs located a medical determination of the presence or absence of UL in exons 2 and 3 of CYP1B1 (Arg368His, Leu432Val, after a medical examination and the diagnosis was confrmed Asp449Asp and Asn453Ser) in an Iranian population [26]. by the gynecologist afliated to the Gynecology Clinic. Par- Their data showed that none of these individual SNPs had ticipants were also asked to complete a brief biodata ques- an association with disease risk; however, they suggested tionnaire about race, age and fbroid status. All participants that the GTAA haplotype may have a protective function in self-identifed as black or Afro-Caribbean [27]. All partici- UL development, as opposed to a causative one. Similarly, pants gave informed consent prior to participating in the Gooden found no association with UL risk when two com- study and the study was approved by the Ethics Committee mon variants (Ala119Ser and Leu432Val) were haplotyped of the University of the West Indies Cave Hill. in women with African or European ancestries [25]. There have been no haplotype studies of the Leu432Val SNP and Primer design UL risk in a Caribbean population, although a protective role was proposed for the recessive allele [G/C] or ­Val432 in Primers used to generate PCR fragments for sequencing a previous study of the Leu432Val SNP in the same popula- were designed using Primer BLAST [33]. The primers tion [27]. CYP1B1F (5′-TGA​AGA​ACC​GCT​GGG​TAT​GG-3′) and Additional risk factors besides genetics such as diet, CYP1B1R (5′-CCA​CAT​TAA​ACA​CCA​AAC​AGGT-3′) for obesity, parity, and hypertension among others have also CYP1B1 (accession # NC_000002.12) were selected from been widely associated with the prevalence of UL [6, 8, a region spanning 779 bp and contained three common SNPs 10, 28]. Women of African ancestry with UL tend to have (Leu432Val, Asp449Asp and Asn453Ser). Figure 1a shows high mean BMIs (> 25 kg/m2) [28] and an increased risk of the distribution of the three SNPs in CYP1B1. UL is observed in obese women [24, 29–31]. A candidate The primers CCDC57F (5′-TTTAGC​ TCT​ TGT​ GGC​ CCC​ ​ SNP rs4247357, located in a large LD block on chromo- TC-3′), and CCDC57R (5′-TCA​ACT​ACA​AGA​CTT​CGA​ some 17q25.3, was implicated in UL susceptibility among CTTCAA​ C-3​ ′) were generated from a 1500 bp region span- a cohort of women with European ancestry [32]. However, ning rs4247357 (Fig. 1b) as well as two other variants iden- this genetic association was not replicated in a larger genome tifed from the African-Caribbean, Barbados population in wide association study in a multiethnic group of diferent the 1000 genomes project [19]. The CCDC57 PCR primers ancestries that did not include Afro-Caribbean women amplifed a 571 bp LD region. [12]. The LD block contains three genes; FASN, Coiled- Coil Domain Containing 57 (CCDC57) and Solute Carrier PCR amplifcation and sequencing Family 16 (SLC16A3). The SNP located in CCDC57 causes a nucleotide change [C/A] in the gene. There are Amplifcations were performed in a Techne Genius thermal no reported haplotype studies of rs4247357 of CCDC57 cycler (Fisher Scientifc, Suwanee, GA, USA). The total and UL risk among blacks or whites. Consequently, in this volume for each reaction mixture was 25 µL containing study we utilized experimentally determined sequence data ~ 200 ng of genomic DNA, 1 µM of primers (Bio-Synthesis to identify whether haplotypes in CYP1B1 and CCDC57 are Inc., Lewisville, TX, USA), 2 mM of dNTP mix, 2 mM linked to UL risk in an Afro-Caribbean female population ­MgCl2, 1 × PCR bufer and 1.25 U Amplitaq Gold 360 in Barbados. DNA polymerase (Applied Biosystems Inc., Foster City, CA, USA) and sterile distilled water. A no template control was used as a negative reaction control. Materials and methods The optimal reaction conditions for CYP1B1 amplifca- tion were as follows: initial denaturation was performed at Participant sampling 95 °C for 2 min followed by 40 cycles at 95 °C for 1 min, 60 °C for 30 s, 72 °C for 30 s and a fnal extension step at A total of 108 blood DNA samples were obtained over a 72 °C for 5 min. Visualization of amplifed fragments was 2-year period (2009–2011), from females who were part of performed on 2% ethidium bromide (EtBr) stained agarose the Barbados Fibroid Study [27]. Each was amplifed by gels. PCR and subsequently sequenced for CYP1B1 and CCDC57. Optimal reaction conditions for CCDC57 were as follows: In each analysis, the study population comprised women initial denaturation was performed at 95 °C for 45 s followed who were being treated at the Gynecological Outpatient by 35 cycles at 95 °C for 45 s, 64 °C for 30 s, 72 °C for 30 s Clinic of the Queen Elizabeth Hospital in Barbados for and a fnal extension at 72 °C for 5 min. Amplifed fragments symptomatic UL as cases, and a non-UL control group were visualized on a 2% agarose gel stained with EtBr.

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Fig. 1 Schematic representation of the distribution of three SNPs is a region of high linkage disequilibrium that spans three genes con- in CYP1B1 and CCDC57. a The SNPs are located in exon 3 of the taining approximately 22 SNPs which have been genotyped in other gene. Leu432Val causes an amino acid change from leucine to valine, populations. Only the three shown above were polymorphic and Asp449Asp is a silent mutation of aspartate and Asn453Ser causes a therefore relevant to the current study change from asparagine to serine. b The 0.5 kb segment of CCDC57

The analysis of CYP1B1 and CCDC57 haplotypes in this Sanger sequencing of CYP1B1 and CCDC57 (GENEWIZ, study was performed by DNA sequencing. All PCR prod- South Plainfeld, NJ, USA) were analyzed using Geneious ucts were sequenced using PCR optimized primers. Prior version 9.0.5 [34]. Genotypes for each analyzed SNP under to sequencing, the PCR amplicons generated were purifed investigation (Table 1) were identifed and coded according using the ChargeSwitch-Pro PCR Clean-Up Kit (Invitrogen, to the International Union of Pure and Applied Chemistry Waltham, MA, USA) as described in the manufacturer’s (IUPAC) ambiguity index. The consensus sequences for instructions. The DNA concentrations of the purifed prod- each gene were mapped to the reference sequences, Homo ucts were assessed by measuring A260 nm values using a sapiens 2 (NC_000002.12) and chromosome Quantus Fluorometer (Promega, Madison, WI, USA). A 17 (NC_000017.11) from the GRCh38.p2. Sequence align- template volume of 10 µL for each reaction was provided ments were obtained by Nucleotide BLAST (BLASTn) [35] for sequencing using the ABI PRISM BigDye Terminator for CYP1B1 and CCDC57. cycle sequencer (GENEWIZ, South Plainfeld, NJ, USA) in A total of 85 and 100 sequences were therefore assembled a 96-well plate. for CYP1B1 and CCDC57, respectively. Haplotypes were reconstructed from unphased data in CYP1B1 for 40 cases Haplotyping with uterine leiomyomas and 45 controls without UL. Sev- eral more persons were enrolled for the subsequent study of The exclusion criteria for data analysis were sequenced sam- CCDC57 and these haplotypes were reconstructed by using ples with poor quality base calling. The ABI chromatograms 43 cases and 57 controls. Haplotype frequency was analyzed (forward and reverse.abi fles), obtained from bidirectional using the Bayesian approach of Stephens et al. (2001).

Table 1 SNP haplotypes of SNP ID Gene Alleles Amino acid change Positiona Minor allele IUPACd CYP1B1 CCDC57 and genes frequency ambiguity code

rs1056836 CYP1B1 [C/G] Leucine to Valine 38071060 0.39 S rs1056837 CYP1B1 [A/C/T] Aspartate to Aspartate 38071007 0.38 Y rs1800440 CYP1B1 [G/A] Asparagine to Serine 38070996 0.09 R rs4247357 CCDC57 [C/A] ndb 82209113 0.40 M rs150468462c CCDC57 [T/C] nd 82209060 0.01 Y rs6502087 CCDC57 [A/G] nd 82208994 0.04 R

a SNP position on chromosome 2 for CYP1B1 and for CCDC57 b No known amino acid change detected for this SNP c The rs150468462 SNP is not found in HapMap but is present in 1000 genome database d The International Union of Pure and Applied Chemistry

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Haplotype analyses of CCDC57 have not previously been Table 2 Allelic frequencies of the GTA and GTC haplotypes in reported, as such, variants including rs4247357 were identi- CYP1B1 and CCDC57 fed in the 1000 genome project [19]. The sample dataset SNP ID Associa- Cases (%) Controls (%) χ2 Pa was then limited to include only SNPs that were common tion allele to our sample population based on the heterozygotes identi- Leu432Val G 93 83 3.29 0.07 fed during assembly to a reference generated from BLASTn Asp449Asp T 83 62 8.60 0.003 [35] and the MUSCLE MSA [36]. The SNPs chosen for Asn453Ser A 99 97 0.80 0.37 analysis were based on their presence in the HapMap and rs6502087 G 94 94 0.01 0.92 1000 Genome Project databases along with their base pair rs150468462 T 98 97 0.24 0.63 changes. Finally, the minor allele frequencies of each SNP rs4247357 C 52 50 0.11 0.75 investigated were obtained from the dbSNP database [37] based on frequency of observation in a larger West African, a P values were assumed statistically signifcant at < 0.05 Yoruba population (Table 1).

Statistical analysis

Chi-squared goodness-of-ft tests and Hardy–Weinberg equi- librium (HWE) were calculated for the controls to assess whether the population was in equilibrium. A post hoc power calculation was conducted to determine a medium efect size (d = 0.5) on the two groups in the two genes. T-tests for two independent groups using the Wilcox Mann–Whit- ney test were computed on a sample size of 85 and 100, for CYP1B1 and CCDC57 respectively, using GPower [38]. The alpha level used was p < 0.05. Haplotype frequencies of the unphased data were calculated using PHASE v.2.1 [39]. Any haplotype with an estimated frequency of < 5% was excluded from association and logistic regression analy- ses. To assess the independent efect of each SNP, logistic regression was used, and a post hoc Bonferroni’s correction test was performed to confrm the associations observed with Fig. 2 CYP1B1 CCDC57 each haplotype and the disease. Pairwise linkage disequi- LD plots for SNPs in and . a An LD plot of three SNPs in CYP1B1. a The bright red square is indicative of com- librium between SNPs and minor allelic associations were plete linkage disequilibrium (D′ = 1) for the Asp variant; the lighter computed in Haploview v.4.2 [40]. shade of red shows high disequilibrium for the Leu variant as well (D′ < 1) and the blue square for the Asn variant shows high disequi- librium when (D′ = 1 and LOD = < 2). b An LD plot of three SNPs in CCDC57. The white square represents high recombination while Results the blue squares indicate D′ = 1 with a LOD score < 2. (Color fgure online) The mean ages for cases and controls were 46.3 y ± 7.23 and 45.4 y ± 10.50, respectively for CYP1B1, and for CCDC57 the mean ages were 42.2 y ± 8.23 and 33.4 y ± 10.42 for the mutation in the Asp449Asp genotype was present in 83% cases and controls, respectively. There was no signifcant of the cases and was substantially lower in the controls at diference between ages of cases and controls in either of 62% and the minor allele T of this SNP was frequently and the haplotypes of the two genes studied (P > 0.05). Post-hoc statistically associated with UL cases (P =0.003) (Table 2). analysis revealed a low power of 0.63 for a medium efect for However, alleles of rs4247357 SNP were equally likely to CYP1BI, but an appropriate power of 0.82 for CCDC57 for be present in both cases and controls in this population and a medium efect on such a sample size. An a priori sample there was no statistical signifcance observed (p > 0.05). size for a larger efect of 132 is required for a similar efect The LD plot of the CYP1B1 SNPs (Fig. 2a) showed that for CYP1B1. Leu432Val and Asp449Asp are in high LD and are unlikely In CYP1B1, the variant Asn453Ser major allele A (99%) to have had a recombination event. The Asn453Ser SNP is had the largest distribution for any SNP with an associated also in high LD however it may not be inherited on the same haplotype. Similar values were observed for the cases car- chromosome as the other two variants. A pairwise compar- rying the Leu432Val minor allele G (Table 2). The silent ison of SNPs associated with CCDC57 (Fig. 2b) showed

1 3 Molecular Biology Reports (2019) 46:3299–3306 3303 that there is a very little LD among these three SNPs and haplotype (odds ratio 3.98, P value = 0.02) showed a sig- rs4247357 shows high recombination. nifcant diference in the population, indicating that it was signifcantly associated with UL risk. In addition, CCG and Haplotype frequencies CTA haplotypes had the lowest frequencies with values of < 5% in the both cases and controls. Furthermore, the esti- The GTA haplotype for CYP1B1 is widely distributed in the mated GTA frequency was higher in the cases than in the population while CTA and CCG haplotypes for CCDC57 controls (82% compared to 61%, respectively). with minor alleles of rs6502087 and rs150468462 are rare. All haplotypes of the SNPs were in HWE in both cases For CYB1B1 (Table 3), only the frequency of the GTA and controls. The Bonferroni correction post hoc was 0.40 and combined haplotype frequencies of the total study popu- lation are shown in Fig. 3. The GCA haplotype frequency Table 3 Haplotype frequency of SNPs in CYP1B1 and CCDC57 was two times higher in the control population than in the among Afro-Caribbean Barbadian women cases and altogether, GTA had the highest frequency in the Haplotype (a/b) Cases Controls Odds ratio (95% CI)d P-valuec sample population (Fig. 3a). For CCDC57 there were no sta- tistically signifcant haplotypes of rs4247357 and associated CCAa​ 0.07 0.12 1.00c – a UL risk in the sample population (Table 3). Only the CTA CCG​ 0.01 0.03 – – a haplotype had more cases (6%) than controls (3%) (Fig. 3b), CTA​ – 0.013 – – a while the haplotypes of CCG, ACG and ATA were present GCA​ 0.10 0.22 1.66 (0.50–5.48) 0.41 a in values lower than 3% in both groups (Table 3). GTA​ 0.82 0.61 3.98 (1.29–12.34) 0.02 CTG​b 0.44 0.44 1.00c – b CCG​ 0.02 0.02 – – Discussion ATG​b 0.48 0.46 1.66 (0.76–3.62) 0.20 ACG​b – 0.01 – – b Uterine fbroids have been established as a complex disease CTA​ 0.06 0.03 – – b involving genetic, environmental and biochemical factors [4, ATA​ 0.001 0.03 – – 10, 13, 28, 41, 42]. Accordingly, although SNPs of CYP1B1 a CYPIB1 genes and CCDC57 have been genotyped for UL risk in African b CCDC57 gene and European ancestral populations [12, 27, 32, 43], there c P values were assumed statistically signifcant at < 0.05 are no studies of haplotypes in Afro-Caribbean populations. d CI confdence interval Therefore, we assessed haplotypes of the two genes CYP1B1

Fig. 3 Haplotype frequencies of CYP1B1 and CCDC57 for cases and controls combined. a CYP1B1 haplotype frequency, b Haplotype frequency of CCDC57

1 3 3304 Molecular Biology Reports (2019) 46:3299–3306 and CCDC57 for their association with UL risk in an Afro- afect the expressed phenotype [51, 52] and as such are use- Caribbean Barbadian population. ful in candidate-gene studies involving complex diseases. Interestingly, when the hormonally relevant CYP1B1 However, studies have shown that populations of greater gene was investigated, the most critical fnding showed that African ancestry tend to have high haplotype diversity but there was an association between the GTA haplotype and UL low LDs in their nuclear genomes [53]. Therefore, nearby risk among this group of Afro-Caribbean females as seen markers close to single variants associated with a particular in Table 3. The three SNPs were also in high LD indicating trait may form haplotypes that are in much higher LD with that they were inherited together and there was very little the functional variant leading to a possible area for further recombination. research [51, 52]. Several genetic association studies have been conducted There was no signifcant association observed for the hap- on SNPs in CYP1B1 and UL risk in diverse populations lotype frequencies of CCDC57 and the disease. Both CTG [26, 44, 45]. Ye et al. studied Leu432Val and Asp449Asp and ATG haplotypes had a relatively equal chance of occur- in a Chinese population and found no association with ring in both cases and controls as seen in Table 3. It was also these SNPs and this disease [44]. Similarly, in the Han observed that the SNPs rs150468462 and rs6502087 were Chinese population with UL there was no association with very rare in this population (< 5%) and as such no haplo- Asp449Asp and UL, although the Leu432Val was protective types with minor alleles (G or C respectively) were evalu- for UL risk [45]. Likewise, there was no association between ated for UL risk. This rarity is confrmed by minor allele UL risk and any of the single polymorphisms investigated frequency reports obtained from dbSNP [37]. The MAF has in an Iranian population [26]. However, it was proposed that been reported as 0.48 for the minor A allele of rs427357 the haplotype GTAA may play a protective role among Ira- compared to 0.04 for minor allele C in the rs6502087 SNP nian women with UL. Previous fndings in the Barbadian and 0.01 for G in the rs150462468 SNP. These approximate female population also indicate a lack of association between frequency values were also observed in the study. The ampli- UL risk and the Leu432Val SNP [27] and this is further fed fragment containing these three SNPs is in a large LD confrmed by the allelic frequency distribution data of the region spanning FASN, SLCA16 and CCDC57, however GTA haplotype in Table 2. However, a closer examination of from the LD plot generated it appears that all three SNPs are each allelic frequency value in Table 2 showed that the Leu- not in LD with each other and as such it validates the lack 432Val and Asn453Ser minor alleles G and T respectively of association observed. In addition, none of these variants were not associated with UL risk, but in the silent mutation have been reported to cause amino acid changes. Additional Asp449Asp, the minor allele T was statistically signifcant. participants were enrolled for CCDC57 analysis and the So, despite any published reports of the importance of the sample size was appropriate with a power of 0.8. However, Asp449Asp SNP and UL risk in other population studies a slightly larger sample pool is required for CYP1B1 for a [26, 44, 46] the current sequence data suggest that the more similar efect. important risk allele for UL among Afro-Caribbean females A previous study of UL risk and SNPs in this population from Barbados is Asp449Asp. also measured both obesity and estradiol levels [54]. Both CYP1B1 is a major enzyme involved in estrogen metabo- of these were signifcantly associated with UL risk, such lism and it has been reported to play a pivotal role in tumor that Afro-Caribbean Barbadian women with UL were shown development by catalyzing the conversion of estrogen to to have BMI’s > 25 ± 8.0 kg/m2 and were of greater risk of 4-hydroxyestradiol which can be converted to genotoxic having high estrogen levels [54]. metabolites [47]. The function of this enzyme is regulated by Caribbean populations, especially persons of Afro- CYP1B1 and as such polymorphisms of that gene, particu- Caribbean descent are genetically mixed [20, 55] and an larly those which cause amino acid changes may lead to dis- admixture study of Barbadians who self-identifed as Black parities in normal estrogen metabolism [47, 48]. It has been showed they were genetically diferent with a 77% African proposed that the combined efects of several sequence vari- ancestry mostly of Yoruba origin, 16% European ancestry ants particularly those which are in high LD as in the case of and 6% Asian [56], when compared to African Americans. the GTA haplotype may be directly responsible for changes Although we did not investigate the Arg49His polymor- in promoter activity or structure, thereby inducing phism nor did we conduct a population structure analysis tumor development [49]. In addition, the haplotypic efects based on ancestry informative markers, we expect that observed in CYP1B1 may be a combined efect of mutations because Afro-Barbadians historically and genetically show resulting in amino acid changes which have a more substan- more African ancestry than Afro-Caribbean blacks in some tial efect on the functioning of protein products compared islands with higher levels of European or Asian ancestry [20, to single amino acid changes [50]. Reports have suggested 55] the genetic variations would be more easily recognized that mutations in a single gene inherited on the cis chromo- here, than where the ethnic stratifcation is less homoge- some can interact to form a ‘super allele’ which can largely neous. Therefore these fndings are compelling because of

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