Abstract
Black women are disproportionately affected by uterine leiomyomata (UL), or fibroids, compared to other racial groups, having a greater lifetime risk of developing UL and an earlier age of diagnosis. In order to elucidate molecular and genetic mechanisms responsible for the increased prevalence and morbidity associated with UL in black women, clinical, pathologic, cytogenetic, and select molecular profiling (MED12 mutation analysis) of 75 self-reported black women undergoing surgical treatment for UL was performed. Our observations are broadly representative of previous cytogenetic studies of UL: karyotypically abnormal tumors were detected in 30.7% of women and 17.4% of analyzed tumors. No notable association was observed between race and increased occurrence of cytogenetic abnormalities that might contribute to any population-specific morbidity or prevalence rate. Our data on MED12 mutation analyses (73.2% of tumors harbored a MED12 mutation) provide additional support for a significant role of MED12 in tumorigenesis. Although the effect of MED12-mediated tumorigenesis appears significant irrespective of race, other genetic events such as the distribution of karyotypic abnormalities appear differently in black women. This case series indicates that presently recognized genetic and molecular characteristics of UL do not appear to explain the increased prevalence and morbidity of UL in black women.
Keywords: Fibroids, race, cytogenetic, molecular, clinical
Graphical Abstracts/Highlights files
Chromosome Displaying Cytogenetic Abnormality | No. of Tumors Displaying Abnormality | Percent of All Abnormal Tumors (%) | Percent of All Tumors Analyzed (%) |
---|---|---|---|
chromosome 6 | 2 | 7.1 | 1.2 |
chromosome 7 | 15 | 53.6 | 9.3 |
chromosome 12 | 5 | 17.9 | 3.1 |
chromosome 14 | 0 | 0 | 0 |
other/complex | 8 | 28.6 | 5.0 |
Point Mutation | No. of Tumors with Mutation | Percent of All Point Mutations (%) | Percent of All Tumors Analyzed (%) |
---|---|---|---|
c.107T>G | 3 | 12 | 7.5 |
c.128A>C | 1 | 4 | 2.5 |
c.130G>A | 2 | 8 | 5.0 |
c.130G>C | 3 | 12 | 7.5 |
c.130G>T | 3 | 12 | 7.5 |
c.131G>A | 9 | 36 | 22.5 |
c.131G>T | 4 | 16 | 10 |
INTRODUCTION
Uterine leiomyomata (UL), more commonly known as fibroids, are benign, clonal smooth muscle tumors of the uterus. UL are the most common pelvic tumor in women. By 50 years of age, 70% of white women and 80% of black women have had at least one fibroid. Severe symptoms, such as abdominal pain, abnormal menstrual bleeding, urinary incontinence, and fertility impairment, develop in 15–30% of these women during their reproductive years. [1] Uterine fibroids are a major public health concern given their high incidence, frequency, and morbidity, and are the primary indication for hysterectomy in the United States. [2–4] The annual direct cost for the clinical management of uterine fibroids in the United States is estimated to be $4.1–$9.4 billion, exclusive of costs attributable to obstetric complications and lost productivity. [5] In addition, they contribute to a decreased quality of life for many women.
Black women are disproportionately affected by UL when compared to other racial groups. [6] In addition to having a greater lifetime cumulative incidence of fibroids, black women are also diagnosed at a younger age. Uterine fibroids are significantly larger at the time of diagnosis in black women compared to white women and are associated with longer phases of sustained growth. [1] Affected black women are also more likely to have multiple fibroids. [7] Even after controlling for known risk factors such as BMI and hypertension, race remains a factor predisposing black women to develop UL, supporting an underlying genetic contribution. [8] In addition to this racial difference in prevalence and morbidity, a genetic component to UL predisposition is substantiated by analyses of twin studies and familial aggregation. Further, cytogenetic and molecular studies have provided evidence of a strong genetic component in the pathobiology of these tumors. [9–10]
Approximately 40% of uterine fibroids are chromosomally abnormal. Consistent, non-random cytogenetic abnormalities account for the majority of these aberrations, notably deletions of 7q, trisomy 12, or rearrangements of 12q15, 6p21, or 10q22. Additional chromosomal abnormalities of varying complexity have been routinely identified. [11–12] Three independent genetic subtypes of UL have emerged with the advent of next-generation sequencing technologies: rearrangements of the gene encoding high-mobility group protein AT-hook 2 (HMGA2); mutations of fumarate hydratase (FH); and mutations of the mediator complex subunit 12 gene (MED12). [11,13–14] HMGA2 is dysregulated in UL with chromosomal rearrangements of 12q15. [15] While mutations of FH at 1q43 are known to encode syndromic forms of UL such as multiple cutaneous and uterine leiomyomata (MCL) and hereditary leiomyomatosis and renal cell cancer (HLRCC), loss of FH may also play a role in the pathogenesis of nonsyndromic UL. [11] Mutations in exon 2 of MED12 have been reported in 50%–70% of UL. [16]
A thorough understanding, however, of molecular and genetic mechanisms responsible for the increased prevalence and morbidity associated with UL among black women is needed to inform future research directions and clinical treatments. The aim of this case series is to present clinical, pathologic, cytogenetic, and select molecular profiles (MED12 mutation analysis) of 75 self-reported black women with UL undergoing surgical treatment at Brigham and Women’s Hospital who enrolled in our ongoing UL-related research studies over a 27-year period.
MATERIALS AND METHODS
Human Subjects Study Approval
Approval for this study was obtained from the Partners Human Research Committee/Institutional Review Board of Partners Healthcare System (Boston, MA).
Study Population
The Center for Uterine Fibroids at Brigham and Women’s Hospital (Boston, MA; www.fibroids.net) has had a longstanding interest in understanding the genetic underpinnings of UL. Women undergoing surgery in the Department of Obstetrics and Gynecology for treatment of UL are consented and enrolled in our research studies. Self-identified black women presented in this report underwent either a myomectomy or hysterectomy at Brigham and Women’s Hospital between 1989 and 2015.
Medical Record Review
Clinical records of all subjects were reviewed. Information such as patient’s age at time of treatment, clinical indication or primary symptom for treatment, tumor size and number, and uterine weight were analyzed.
Tissue Handling and Cytogenetic Analysis
Samples of UL and matched myometrium were collected during or immediately following surgery. For subjects with multiple fibroids, the largest fibroids were selected for analysis. In instances where a minimally invasive morcellation procedure was performed and delineation of individual fibroids was not possible, only a single piece of tissue was selected for study. Tissue for DNA isolation was frozen and stored at −80°C. Tissue for cell culture and cytogenetic analysis was transferred to Hank’s balanced salt solution. From tissue samples collected in Hank’s, cell cultures were established as previously described, and standard GTG-banded karyotyping was performed. [12] Formalin-fixed, paraffin-embedded tissue blocks were obtained for histopathologic analysis and confirmation of UL diagnosis.
DNA Isolation and MED12 Mutation Analysis
DNA was isolated from frozen tissue samples using the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). Using previously reported primer sequences, the desired DNA fragment, exon 2 of MED12, was amplified with Invitrogen Platinum Taq DNA Polymerase (Invitrogen, Carlsbad, CA). [16] Subsequently, PCR products were separated by agarose gel electrophoresis. DNA fragments were extracted using the Qiagen Gel Extraction Kit. DNA sequencing was then performed on an Applied Biosystems 3730xl DNA Analyzer (Applied Biosystems, Waltham, MA) using forward and reverse PCR primers. Sequence chromatographs were analyzed using Geospiza’s FinchTV software (Geospiza Inc., Seattle, WA).
RESULTS
Clinical Evaluation
Our study group consisted of 75 self-reported black women with a confirmed diagnosis of UL. All patients with symptomatic uterine fibroids and a supporting physical examination underwent further ultrasonographic evaluation to confirm UL diagnosis as well as to define the location, size and number of fibroids. Mean age at the time of surgical treatment was 39.5 years (median 39, range 28–57). Forty-one women underwent a myomectomy (36 abdominal myomectomies, five laparoscopic myomectomies), and 34 underwent a hysterectomy (six supracervical hysterectomies, 27 total abdominal hysterectomies, one total vaginal hysterectomy). Fourteen women undergoing hysterectomy and two undergoing myomectomy had either a concurrent unilateral or bilateral salpingo-oophorectomy. Of note, our data on surgical procedures are demonstrative of broader trends in surgical management of UL over the past few decades toward more minimally invasive and uterine-conserving procedures. [17]
The primary indication or symptom prompting medical or surgical intervention was abnormally heavy or prolonged menstrual bleeding, or menorrhagia, with 40 women (53.3%) reporting this symptom. Twenty-eight women (37.3%) reported pelvic pain or pressure. Twelve women (16%) were undergoing surgical intervention in response to fertility complications. Ten women (7.5%) reported urinary impairment such as incontinence. Other indications included abdominal pain, increased abdominal girth, or a pelvic/abdominal mass. Twenty-one women (28%) reported two or more of these symptoms.
Clinical management of UL frequently involves treatment with gonadotropin-releasing hormone agonists (GnRHa) to provide relief from excessive vaginal bleeding and to reduce uterine volume and fibroid size prior to surgery. [18] Sixteen women (21.3%) had received leuprolide, a GnRHa, prior to surgical intervention.
Hysterectomy remains the only essentially curative treatment for UL. Between 10% and 25% of women who have a myomectomy will require additional surgical intervention for recurrent fibroids. [19] In our study, 13 women (17.3%) had undergone previous surgical intervention before their procedure at Brigham and Women’s Hospital. The average time since most recent surgical intervention for UL was 44.1 months (median 48, range 3.5–84) across the cohort. Eleven women (14.7%) who underwent a myomectomy at the time of the study required future surgical intervention. The average time until subsequent intervention was 74.5 months (median 62.8, range 1.63–168). Five women who had undergone a previous surgical intervention opted for a hysterectomy. Of note, one woman underwent three hysteroscopic myomectomies before undergoing a total abdominal hysterectomy with bilateral salpingo-oophorectomy as part of a treatment plan for uterine adenosarcoma. Fully comprehensive follow-up was not possible for all subjects given that our access to medical record was restricted to only those women who continued their care in the Partners Healthcare System network.
Pathologic Evaluation
For women undergoing hysterectomy, the mean uterine weight was 591.9 grams (median 401, range 66–4678). The mean uterine diameters were 12.8 × 10.1 × 7.3 centimeters. Across the entire cohort, the mean number of tumors per woman indicated by gross pathologic examination was 18.2 (median 11, range 1–109). The exact number of tumors present for 29 women was unobtainable due to either absent information or a record only of “multiple” fibroids. The mean value of the greatest tumor dimension was 7.8 centimeters (median 6.5, range 0.5–17). Reported tumor locations were subserosal (51 women), intramural (49), submucosal (31), and fundal (15). Fourteen women had pedunculated fibroids. Fifty-three women had tumors in two or more locations. Twenty-nine women had tumors in three or more locations.
A more thorough histopathologic analysis was performed for 40 cases. Thirty-three cases were diagnosed as usual type UL, two as usual type UL with hyalinization, one as usual type UL with myxoid changes, one as usual type UL with plexiform changes, and three as cellular/focally cellular UL. Additionally, pathologic evaluation of the tumors indicated 13 cases of adenomyosis. Twenty-eight cases had accompanying chronic cervicitis, cervical squamous metaplasia, mucous cysts, or parakeratosis.
Cytogenetic Evaluation
One hundred sixty-one individual tumors (an average of ~two tumors per case) were successfully karyotyped. Twenty-eight of these tumors (17.4% of all tumors analyzed) from 23 cases (30.7%) were karyotypically abnormal. In the 47 cases in which more than one tumor was successfully karyotyped, 15 cases (31.9%) had tumors with both abnormal and normal karyotypes. Of the 16 subjects reported to have received leuprolide treatment, eight subjects (50%) had a cytogenetic abnormality identified in their tumors. Table 1 provides information on all tumors which had an abnormal karyotype or a recognized constitutional variant. Abnormalities are arranged by the most common cytogenetic rearrangements associated with UL in Table 2. [20] The most frequent cytogenetic abnormality observed was an interstitial deletion in the long arm of chromosome 7.
Table 1.
Subject | Age at Diagnosis (years) | No. of Fibroids | Case Number | Karyotype | Cytogenetic Abnormality | MED12 Mutation (if analyzed) |
---|---|---|---|---|---|---|
| ||||||
UL02 | 48 | multiple | ST91-328 | 43,X,-X,del(1)(p32->pter),−6,del(7)(q22q32), −19[16]/46,XX[7] | complex | |
| ||||||
UL03 | 46 | 12 | ST91-314 | 46,X,r(X)[8]/46,XX[13] | ring | |
ST91-315 | 46,XX | |||||
| ||||||
UL07 | 46 | 5 | ST93-248* | 46,XX,inv(9) | ||
ST93-249* | 46,XX,inv(9) | |||||
| ||||||
UL09 | 49 | multiple | ST99-045 | 46,XX,del(7)(q22q32)[8]/46,XX[2] | del(7) | |
ST99-046 | 46,XX,del(7)(?q11?q11.2)[2]/46,XX[4] | del(7) | ||||
ST99-048 | 46,XX | |||||
ST99-049 | 46,XX | |||||
| ||||||
UL15 | 38 | 6 | ST89-185 | 46,XX | ||
ST89-186 | 50,XX,dup(12)(q14->q24),+21,+21,+21,+21[1]/46,XX[65] | complex | ||||
| ||||||
UL16 | 38 | multiple | ST99-735 | 46,XX | ||
ST99-736 | 46,XX | c.107T>G | ||||
ST99-737 | 46,XX,t(6;10)(p21;q22) | 6p rea | c.130G>A | |||
ST99-738 | 46,XX | |||||
| ||||||
UL19 | 40 | 19 | ST93-470 | 46,XX,del(7)(q22q32)[19]/46,XX[1] | del(7) | |
ST93-471 | 46,XX | |||||
| ||||||
UL21 | 46 | multiple | ST99-122 | 45,XX,−22[6]/46,XX[2] | monosomy 22 | |
ST99-123 | 46,XX,del(7)(q22q32)[11] | del(7) | ||||
| ||||||
UL23 | 40 | multiple | ST94-190* | 46,XX,9qh+ | ||
ST94-191* | 46,XX,9qh+ | |||||
ST94-192* | 46,XX,9qh+ | |||||
ST94-193 | 46,XX,del(7)(q21.1q34),9qh+[3]/46,XX,9qh+[17] | del(7) | ||||
| ||||||
UL25 | 38 | 3 | ST93-054 | 46,XX | ||
ST93-055 | 45,XX,t(1;7)(q31;q22),−2,−3,−6,add(11)(q21),−18,+mar1,+mar2,+mar3 | complex | ||||
ST93-056 | 46,XX | |||||
| ||||||
UL26 | 39 | 21 | ST95-479 | 46,XX,del(7)(q22q32)[6]/46,XX,?inv(7)(q21q22)[2]/46,XX[10] | del(7) | |
ST95-480 | 46,XX | |||||
| ||||||
UL27 | 38 | multiple | ST95-520* | 46,XX,inv(9) | ||
ST95-521* | 46,XX,inv(9) | |||||
ST95-522* | 46,XX,inv(9) | |||||
| ||||||
UL32 | 38 | 5 | ST96-437 | 46,XX,del(7)(q22q32)[1]/46,XX[14] | del(7) | |
| ||||||
UL37 | 36 | multiple | ST95-364 | 46,XX | ||
ST95-365 | 46,XX,t(1;13)(p22;q14),−11,+mar?(11pter- >q12:)[8]/46,XX[2] | complex | ||||
ST95-366 | 46,XX | |||||
| ||||||
UL40 | 33 | 5 | ST93-731 | 46,XX | ||
ST93-732 | 46,XX | |||||
ST93-733 | 46,XX,del(7)(q22q32)[13]/46,XX[2] | del(7) | ||||
| ||||||
UL41 | 39 | multiple | ST00-025 | 45,XX,r(?13),−22 | ring | |
ST00-026 | 46,XX | c.107T>G | ||||
| ||||||
UL42 | 42 | multiple | ST02-278 | 46,XX,t(10;12)(q23;q15) | t(10;12) | c.131G>A |
| ||||||
UL48 | 33 | multiple | ST96-481 | 46,XX | ||
ST96-482 | 46,XX,del(7)(q22q32)[10]/46,XX[10] | del(7) | ||||
| ||||||
UL51 | 43 | 14 | ST09-009 | 46,XX | ||
ST09-010 | 45,X,-X,t(6;16)(q13;q24),del(7)(q22) | del(7) | ||||
ST09-011 | 46,XX | c.116-154del39 | ||||
ST09-012 | 46,XX | |||||
| ||||||
UL53 | 40 | 3 | ST08-014 | 46,XX,del(7)(q22q23) | del(7) | c.122_156del35 |
ST08-015 | 46,XX,t(1;6)(p36;p21)[8]/46,XX[13] | t(1;6) | c.130G>C | |||
| ||||||
UL54 | 34 | multiple | ST01-901 | 45,X,-X,r(1),t(4;6)(p16;q21),der(12)inv(12)(p13q11)ins(12)t(12;14)(q15;q21q24),del(14)(q21q24) | complex | wt |
| ||||||
UL56 | 39 | 15 | ST08-021 | 46,XX | ||
ST08-022 | 46,XX | |||||
ST08-023 | 46,XX,del(7)(q11q31) | del(7) | c.131G>A | |||
| ||||||
UL57 | 34 | 11 | ST05-001F1* | 46,XX,9qh+,22pstk+ | ||
ST05-001F2* | 46,XX,9qh+,22pstk+,+mar[3]/46,XX,9qh+,22pstk+[7] | |||||
| ||||||
UL61 | 37 | 9 | ST09-007 | 47,XX,+12 | trisomy 12 | wt |
| ||||||
UL62 | 35 | 13 | ST08-017* | 46,XX,inv(19) | c.131G>A | |
ST08-018* | 46,XX,inv(19) | wt | ||||
ST08-019* | 46,XX,inv(19) | wt | ||||
| ||||||
UL64 | 29 | 2 | ST02-919 | 46,XX,t(1;14;12)(q21;q24;q15) | t(1;14;12) | |
ST02-920 | 47,XX,+add(1)(p13),add(12)(q15) | complex | ||||
| ||||||
UL69 | 29 | 3 | ST09-036 | 46,XX | c.131G>A | |
ST09-037 | 46,XX,+12 | trisomy 12 | ||||
ST09-038 | 46,XX,del(7) | del(7) |
inv(9), 9qh+, inv(19) and 22pstk+ interpreted as constitutional variants not of clinical significance
Table 2.
Chromosome Displaying Cytogenetic Abnormality | No. of Tumors Displaying Abnormality | Percent of All Abnormal Tumors (%) | Percent of All Tumors Analyzed (%) |
---|---|---|---|
chromosome 6 | 2 | 7.1 | 1.2 |
chromosome 7 | 15 | 53.6 | 9.3 |
chromosome 12 | 5 | 17.9 | 3.1 |
chromosome 14 | 0 | 0 | 0 |
other/complex | 8 | 28.6 | 5.0 |
Molecular Evaluation
Forty karyotyped tumors from 28 cases were analyzed for the presence of mutations in exon 2 of MED12. Selection of a tumor for MED12 mutation analysis was based solely on availability of tissue. Mutations were found in 30 (75%) of tumors. The most common mutations were heterozygous point mutations in codons 43 and 44 (25 tumors, 62.5% of tumors analyzed). There were also five cases with deletions spanning exon 2. Tumors from five subjects who received leuprolide treatment were analyzed for presence of a MED12 mutation; three subjects had tumors containing MED12 mutations (60%) occurring exclusively in the coding region of exon 2. Additionally, Mäkinen et al. [16] performed whole exome sequencing and reported mutations only in exon 2, and no mutations in noncoding regions adjacent to MED12. Only two tumors had rearrangements involving 12q15, one of which had a MED12 mutation. Detailed information on MED12 analysis is provided in Table 3, and the distribution of the different MED12 point mutations is shown in Table 4.
Table 3.
Subject | Age at Diagnosis (years) | No. of Fibroids | Greatest Fibroid Dimension (cm) | Case Number | MED12 Mutation | Karyotype |
---|---|---|---|---|---|---|
| ||||||
UL16 | 38 | multiple | 6.5 | ST99-736 | c.107T>G, p.L36R | 46,XX |
ST99-737 | c.130G>A, p.G44S | 6p rea | ||||
| ||||||
UL36 | 42 | multiple | 12.5 | ST00-399 | c.IVS1-9_140del50 | 46,XX |
| ||||||
UL38 | 42 | 24 | 4.5 | ST02-020 | c.107T>G, p.L36R | 46,XX |
ST02-021 | c.130G>T, p.G44C | 46,XX | ||||
| ||||||
UL41 | 39 | multiple | 5.8 | ST00-026 | c.107T>G, p.L36R | 46,XX |
| ||||||
UL42 | 42 | multiple | 12 | ST02-278 | c.131G>A, p.G44D | t(10;12) |
| ||||||
UL46 | 50 | 33 | 7.5 | ST11-002T2 | c.130G>C, p.G44R | 46,XX |
| ||||||
UL50 | 45 | 8 | 12.5 | ST09-029 | c.130G>T, p.G44C | 46,XX |
| ||||||
UL51 | 43 | 14 | 15 | ST09-009 | c.143_168del26, p.Q48_H56 | 46,XX |
ST09-010 | c. 31G>A, p.G44D | del(7)* | ||||
ST09-011 | c.116-154del38, p.L39_V51 | 46,XX | ||||
| ||||||
UL52 | 44 | 19 | 5 | ST09-024 | c.130G>T, p.G44C | 46,XX |
| ||||||
UL53 | 40 | 3 | 9.5 | ST08-014 | c.122_156del35, p.V41_S52 | del(7) |
ST08-015 | c.130G>C, p.G44R | t(1;6) | ||||
| ||||||
UL54 | 34 | multiple | 10 | ST01-901 | wt | complex |
| ||||||
UL55 | 33 | 28 | 8 | ST02-137 | c.130G>A, p.G44S | 46,XX |
ST02-138 | c.130G>C, p.G44R | 46,XX | ||||
| ||||||
UL56 | 39 | 15 | 9 | ST08-023 | c.131G>A, p.G44D | del(7) |
| ||||||
UL57 | 34 | 11 | 10 | ST05-001F1 | c.131G>A, p.G44D | 46,XX,9qh+,22pstk+** |
| ||||||
UL58 | 37 | 3 | 5 | ST08-025 | c.131G>T, p.G44V | 46,XX |
| ||||||
UL60 | 40 | 6 | 8 | ST12-006T1 | c.131G>T, p.G44V | 46,XX |
ST12-006T2 | wt | 46,XX | ||||
| ||||||
UL61 | 37 | 9 | 5 | ST09-007 | wt | trisomy 12 |
| ||||||
UL62 | 35 | 13 | 17 | ST08-017 | c.131G>A, p.G44D | inv(19)** |
ST08-019 | wt | inv(19)** | ||||
| ||||||
UL63 | 35 | 9 | 10 | ST09-002 | wt | 46,XX |
| ||||||
UL64 | 29 | 2 | 12.5 | ST02-919 | wt | t(1;14;12) |
| ||||||
UL65 | 35 | 9 | 7.8 | ST09-020 | wt | 46,XX |
| ||||||
UL66 | 38 | 41 | 15 | ST12-011T1 | c.128A>C, p.Q43P | 46,XX |
ST12-011T2 | c.90_101del12, p.F30_D34 | 46,XX | ||||
| ||||||
UL67 | 35 | 15 | 10.5 | ST12-002T1 | c.131G>T, p.G44V | 46,XX |
ST12-002T2 | c.131G>A, p.G44D | 46,XX | ||||
| ||||||
UL68 | 33 | 13 | 12 | ST13-001T2 | c.131G>T, p.G44V | 46,XX |
ST13-001T3 | wt | 46,XX | ||||
| ||||||
UL69 | 29 | 3 | 12 | ST09-036 | c.131G>A, p.G44D | 46,XX |
| ||||||
UL70 | 30 | 105 | 16.5 | ST12-001T1 | wt | 46,XX |
| ||||||
UL71 | 28 | 7 | 12 | ST12-003T1 | c.131G>A, p.G44D | 46,XX |
| ||||||
UL73 | 35 | 11 | 3.5 | ST11-001T1 | c.131G>A, p.G44D | 46,XX |
ST11-001T2 | wt | 46,XX |
del(7) indicates the characteristic interstitial deletions seen in UL in the long arm of chromosome 7 with various breakpoints
inv(9), 9qh+, inv(19), 22pstk+ interpreted as constitutional variants not of clinical significance
Table 4.
Point Mutation | No. of Tumors with Mutation | Percent of All Point Mutations (%) | Percent of All Tumors Analyzed (%) |
---|---|---|---|
c.107T>G | 3 | 12 | 7.5 |
c.128A>C | 1 | 4 | 2.5 |
c.130G>A | 2 | 8 | 5.0 |
c.130G>C | 3 | 12 | 7.5 |
c.130G>T | 3 | 12 | 7.5 |
c.131G>A | 9 | 36 | 22.5 |
c.131G>T | 4 | 16 | 10 |
DISCUSSION
The impact of ancestral origin in the biology of uterine leiomyoma is not well understood, although it is well recognized as a risk factor. In regard to genetic studies, large-scale genome-wide association studies and exome sequencing analyses, efforts, and discoveries have been focused on populations of European and Asian ancestry. [10,16,21] There is evidence, however, of racial differences for fibroid development on a molecular level, a discovery that may help guide treatment. [22–24] The information presented in this case series seeks to contribute to a greater understanding of fibroid development on a genetic and molecular level in a disproportionately affected population. In 2013, Moorman et al. [25] published a study on the pathologic and epidemiologic risk factors for UL in black and white women undergoing pre-menopausal hysterectomy. Their analysis of 225 black and 135 white women yielded similar results to our study. However, the study described herein finds a greater mean number of fibroids per patient (18.2 versus 9.9) as well as a greater uterine weight (591.9 g versus 477). [25] A 1996 study by Kjerulff et al. [26] with 409 black women reported a mean uterine weight of 420.8 g. A possible explanation for a seemingly more severe clinical presentation in our study group may be due to the nature of the gynecologic surgical practice at Brigham and Women’s Hospital being primarily referral based and, therefore, a selection bias toward more severe and complex clinical presentations.
Limited published data exist on the cytogenetic abnormalities associated with fibroids specifically in black women. Multiple separate analyses, using predominantly white or non-population-specific cohorts, have demonstrated that approximately 40% of UL tumors are cytogenetically abnormal. [27–29] Thirty-one percent of women in our study had fibroids with cytogenetic abnormalities, while 17.4% of all analyzed tumors were cytogenetically abnormal. Therefore, our cohort broadly resembles previous UL cytogenetic studies with regard to rate of cytogenetic abnormality. More specifically, our data are not supportive of an association between an increased rate of cytogenetic abnormality and race that may contribute to any population-specific morbidity or prevalence. However, one of the most common cytogenetic rearrangements associated with UL, t(12;14), typically found in 20% of all chromosomally abnormal cases was present in only three cases (10.7% of abnormal tumors). Of interest, the distribution of numbers of CT repeats in the 5′ UTR of HMGA2 at 12q15 is strikingly different in black and white women. [30–31] The number of CT repeats (n=27) has been associated with a predisposition to develop UL in a study of white women, and also associated with short stature in this study. [31] Although it remains to be proven, the presence of the 27 CT repeats may potentially mediate t(12;14) and underlie the differential distributions of t(12;14) in black and white women. [31] An enrichment of tumors with deletions of segments of chromosome 7 was observed with 53.6% of all abnormal tumors harboring this aberration. This deletion is reportedly found in 17% of all chromosomally abnormal cases. [32] The other types of abnormalities observed in our cohort are largely consistent with previous studies. [11, 29] However, six tumors (21.4% of abnormal tumors) were observed with very complex cytogenetic abnormalities, and may indicate regions of the genome harboring other genes with pathogenetic variants in the biology of UL.
Since Mäkinen et al. [16] published on the presence of various somatic mutations in exon 2 of MED12 in 2011, numerous mutation analysis studies have been performed in a variety of study populations. [33–38] Mäkinen et al. [39] also published on a separate cohort of 18 women (28 UL) from South Africa identified as “black South African” or “coloured” in 2013. Their mutation rate of 50% of tumors (versus 75% in our group) suggests that MED12 mutations are not substantially more common in black women given that the suggested prevalence of MED12 mutation has ranged from 50% to 70%. [39] Our data provide supportive evidence of a significant role of MED12 in tumorigenesis, irrespective of race or ethnicity.
When our data on MED12 mutations are analyzed in the context of cytogenetic abnormalities, particularly t(12;14), and vice versa, no clear pattern or trend emerges. Of note, however, is the mosaicism among tumors in regard to MED12 mutations belonging to the same case. In 2012, Markowski et al. [20] stratified UL MED12 mutations by cytogenetic subgroup in a German population of 50 patients (80 UL). Our comparatively modest sample size of 28 patients (40 UL) analyzed for MED12 mutation makes any strongly substantiated claims difficult. However, the results from our cohort largely track those of Markowski et al. [20] who reported that 21.9% of their cytogenetically abnormal and 82.6% of their cytogenetically normal tumors displayed MED12 mutations. Of the nine cytogenetically abnormal tumors in our cohort, 66.7% displayed MED12 mutations, and 77.4% of the cytogenetically normal tumors analyzed displayed MED12 mutations.
MED12 is a 45-exon gene on chromosome Xq13 that encodes a single subunit of the Mediator multiprotein complex. [40] Mediator plays critical regulatory roles in multiple, global transcriptional processes, including assembly, initiation, and elongation of the RNA polymerase II at and across gene bodies. MED12 regulates activation of CDK8 kinase by bridging the protein-protein interaction between Cyclin C-CDK8 to the core of the Mediator complex. This interaction acts as a functional switch for alternating between inclusion and exclusion of Cyclin C-CDK8 in the body of the Mediator multiprotein complex, as it corresponds to the subcomplex transitioning between transcriptionally active and repressive states. [40–49] Mutations in exon 2 of MED12 appear to disrupt critical interactions between Cyclin C and CDK8, possibly dysregulating the Mediator complex altogether in a high frequency (~70%) of (UL). [20,50] High frequency, driver mutations located in exon 2 of MED12 are hypothesized to decrease Mediator-associated CDK8 activity at a functional regulatory switch, causing alterations in the global transcriptional programs of UL. [14, 50]
Additional genomic analyses are warranted to determine the landscape that differentiates this gynecologic disorder in women of different ethnic groups. Specifically, advances in next generation sequencing technologies, such as exome and whole genome sequencing, as well as SNP arrays can be used to identify somatic copy number variations in UL tumors. [51–52] Copy number variation arrays can also be used to identify small genomic imbalances and chromothripsis in UL tumors. [53–55] Furthermore, the inclusion of more comprehensively profiled cases of UL in black women is necessary to add sufficient power and significance to our findings. Nonetheless, this case series demonstrates that currently identified genetic and molecular characteristics of UL do not apparently account for the increased prevalence and morbidity of UL in black women.
Acknowledgments
Study data were collected and managed using REDCap electronic data capture tools hosted at Partners Healthcare. [56]
FUNDING
This study was supported by the National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health and Human Development (R01 HD06053001 to C.C.M.).
Footnotes
This study was conducted in Boston, MA.
The authors report no conflict of interest.
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References
- 1.Bulun SE. Uterine fibroids. N Engl J Med. 2013;369:1344–55. doi: 10.1056/NEJMra1209993. [DOI] [PubMed] [Google Scholar]
- 2.Buttram VC, Reiter RC. Uterine leiomyomata: etiology, symptomatology, and management. Fertil Steril. 1981;36:433–45. doi: 10.1016/s0015-0282(16)45789-4. [DOI] [PubMed] [Google Scholar]
- 3.Stewart EA. Clinical practice. Uterine fibroids New Engl J Med. 2015;372:1646–55. doi: 10.1056/NEJMcp1411029. [DOI] [PubMed] [Google Scholar]
- 4.Lepine LA, Hillis SD, Marchbanks PA, Koonin LM, Morrow B, Kieke BA, et al. Hysterectomy surveillance--United States, 1980–1993. Mor Mortal Wkly Rep CDC Surveill Summ. 1997;46:1–15. [PubMed] [Google Scholar]
- 5.Cardozo ER, Clark AD, Banks NK, Henne MB, Stegmann BJ, Segars JH. The estimated annual cost of uterine leiomyomata in the United States. Am J Obstet Gynecol. 2012;206:211e1, 9. doi: 10.1016/j.ajog.2011.12.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Eltoukhi HM, Modi MN, Weston M, Armstrong AY, Stewart EA. The health disparities of uterine fibroid tumors for African American women: a public health issue. Am J Obstet Gynecol. 2014;210:194–9. doi: 10.1016/j.ajog.2013.08.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Huyck KL, Panhuysen CI, Cuenco KT, Zhang J, Goldhammer MS, Jones ES, et al. The impact of race as a risk factor for symptom severity and age at diagnosis of uterine leiomyomata among affected sisters. Am J Obstet Gynecol. 2008;198:168e1, 9. doi: 10.1016/j.ajog.2007.05.038. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Laughlin SK, Baird DD, Savitz DA, Herring AH, Hartmann KE. Prevalence of uterine leiomyomas in the first trimester of pregnancy: an ultrasound-screening study. Obstet Gynecol. 2009;113:630–5. doi: 10.1097/AOG.0b013e318197bbaf. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Wise LA, Ruiz-Narvaez EA, Palmer JR, Cozier YC, Tandon A, Patterson N, et al. African ancestry and genetic risk for uterine leiomyomata. Am J Epidemiol. 2012;176:1159–68. doi: 10.1093/aje/kws276. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Eggert SL, Huyck KL, Somasundaram P, Kavalla R, Stewart EA, Lu AT, et al. Genome-wide linkage and association analyses implicate FASN in predisposition to uterine leiomyomata. Am J Hum Genet. 2012;91:621–8. doi: 10.1016/j.ajhg.2012.08.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Hodge JC, Morton CC. Genetic heterogeneity among uterine leiomyomata: insights into malignant progression. Hum Mol Genet. 2007;16(1):R7–13. doi: 10.1093/hmg/ddm043. [DOI] [PubMed] [Google Scholar]
- 12.Rein MS, Friedman AJ, Barbieri RL, Pavelka K, Fletcher JA, Morton CC. Cytogenetic abnormalities in uterine leiomyomata. Obstet Gynecol. 1991;77:923–6. [PubMed] [Google Scholar]
- 13.Bertsch E, Qiang W, Zhang Q, Espona-Fiedler M, Druschitz S, Liu Y, et al. MED12 and HMGA2 mutations: two independent genetic events in uterine leiomyoma and leiomyosarcoma. Mod Pathol. 2014;27:1144–53. doi: 10.1038/modpathol.2013.243. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Mehine M, Kaasinen E, Mäkinen N, Katainen R, Kampjarvi K, Pitkanen E, et al. Characterization of uterine leiomyomas by whole-genome sequencing. N Engl J Med. 2013;369:43–53. doi: 10.1056/NEJMoa1302736. [DOI] [PubMed] [Google Scholar]
- 15.Gattas GJF, Quade BJ, Nowak RA, Morton CC. HMGIC expression in human adult and fetal tissues and in uterine leiomyomata. Genes Chromosomes Cancer. 1999;25:316–22. [PubMed] [Google Scholar]
- 16.Mäkinen N, Mehine M, Tolvanen J, Kaasinen E, Li Y, Lehtonen HJ, et al. MED12, the mediator complex subunit 12 gene, is mutated at high frequency in uterine leiomyomas. Science. 2011;334:252–5. doi: 10.1126/science.1208930. [DOI] [PubMed] [Google Scholar]
- 17.Jacobson GF, Shaber RE, Armstrong MA, Hung YY. Changes in rates of hysterectomy and uterine conserving procedures for treatment of uterine leiomyoma. Am J Obstet Gynecol. 2007;196:601e1–5. doi: 10.1016/j.ajog.2007.03.009. discussion e5–6. [DOI] [PubMed] [Google Scholar]
- 18.Zullo F, Pellicano M, De Stefano R, Zupi E, Mastrantonio P. A prospective randomized study to evaluate leuprolide acetate treatment before laparoscopic myomectomy: efficacy and ultrasonographic predictors. Am J Obstet Gynecol. 1998;178:108–12. doi: 10.1016/s0002-9378(98)70635-0. [DOI] [PubMed] [Google Scholar]
- 19.Jacoby VL, Jacoby A, Learman LA, Schembri M, Gregorich SE, Jackson R, et al. Use of medical, surgical and complementary treatments among women with fibroids. Eur J of Obstetrics, Gynecology, and Reproductive Biology. 2014;182:220–5. doi: 10.1016/j.ejogrb.2014.09.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Markowski DN, Bartnitzke S, Loning T, Drieschner N, Helmke BM, Bullerdiek J. MED12 mutations in uterine fibroids--their relationship to cytogenetic subgroups. Int J Cancer. 2012;131:1528–36. doi: 10.1002/ijc.27424. [DOI] [PubMed] [Google Scholar]
- 21.Cha PC, Takahashi A, Hosono N, Low SK, Kamatani N, Kubo M, et al. A genome-wide association study identifies three loci associated with susceptibility to uterine fibroids. Nat Genet. 2011;43:447–50. doi: 10.1038/ng.805. [DOI] [PubMed] [Google Scholar]
- 22.Al-Hendy A, Salama SA. Ethnic distribution of estrogen receptor-alpha polymorphism is associated with a higher prevalence of uterine leiomyomas in black Americans. Fertil Steril. 2006;86:686–93. doi: 10.1016/j.fertnstert.2006.01.052. [DOI] [PubMed] [Google Scholar]
- 23.Ishikawa H, Reierstad S, Demura M, Rademaker AW, Kasai T, Inoue M, et al. High aromatase expression in uterine leiomyoma tissues of African-American women. J Clin Endocrinol Metab. 2009;94:1752–6. doi: 10.1210/jc.2008-2327. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Pan Q, Luo X, Chegini N. Genomic and proteomic profiling I: leiomyomas in African Americans and Caucasians. Reproductive biology and endocrinology : RB&E. 2007;5:34. doi: 10.1186/1477-7827-5-34. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Moorman PG, Leppert P, Myers ER, Wang F. Comparison of characteristics of fibroids in African American and white women undergoing premenopausal hysterectomy. Fertil Steril. 2013;99:768–76. e1. doi: 10.1016/j.fertnstert.2012.10.039. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Kjerulff KH, Langenberg P, Seidman JD, Stolley PD, Guzinski GM. Uterine leiomyomas. Racial differences in severity, symptoms and age at diagnosis. J Reprod Med. 1996;41:483–90. [PubMed] [Google Scholar]
- 27.Gross KL, Neskey DM, Manchanda N, Weremowicz S, Kleinman MS, Nowak RA, et al. HMGA2 expression in uterine leiomyomata and myometrium: quantitative analysis and tissue culture studies. Genes Chromosomes Cancer. 2003;38:68–79. doi: 10.1002/gcc.10240. [DOI] [PubMed] [Google Scholar]
- 28.Nilbert M, Heim S. Uterine leiomyoma cytogenetics. Genes Chromosomes Cancer. 1990;2:3–13. doi: 10.1002/gcc.2870020103. [DOI] [PubMed] [Google Scholar]
- 29.Vanni R, Lecca U, Faa G. Uterine leiomyoma cytogenetics II: report of forty cases. Cancer Genet Cytogenet. 1991;53:247–56. doi: 10.1016/0165-4608(91)90101-y. [DOI] [PubMed] [Google Scholar]
- 30.Ishwad CS, Shriver MD, Lassige DM, Ferrell RE. The high mobility group I-C gene (HMGI-C): polymorphism and genetic localization. Hum Genet. 1997;99:103–5. doi: 10.1007/s004390050320. [DOI] [PubMed] [Google Scholar]
- 31.Hodge JC, Cuenco KT, Huyck KL, Somasundaram P, Panhuysen CI, Stewart EA, et al. Uterine leiomyomata and decreased height: a common HMGA2 predisposition allele. Hum Genet. 2009;125:257–63. doi: 10.1007/s00439-008-0621-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Lynch AM, Morton CC. Uterus: leiomyoma. Atlas Genet Cytogenet Oncol Haematol. 2008;12(1):68–73. [Google Scholar]
- 33.Halder SK, Laknaur A, Miller J, Layman LC, Diamond M, Al-Hendy A. Novel MED12 gene somatic mutations in women from the Southern United States with symptomatic uterine fibroids. Molecular genetics and genomics. 2015;290:505–11. doi: 10.1007/s00438-014-0938-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.McGuire MM, Yatsenko A, Hoffner L, Jones M, Surti U, Rajkovic A. Whole exome sequencing in a random sample of North American women with leiomyomas identifies MED12 mutations in majority of uterine leiomyomas. PLoS One. 2012;7:e33251. doi: 10.1371/journal.pone.0033251. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Mäkinen N, Vahteristo P, Kampjarvi K, Arola J, Butzow R, Aaltonen LA. MED12 exon 2 mutations in histopathological uterine leiomyoma variants. Eur J Hum Genet. 2013;21:1300–3. doi: 10.1038/ejhg.2013.33. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Osinovskaya NS, Ivashchenko TE, Dolinskii AK, Sultanov IY, Ghimbovschi S, Hoffman E, et al. MED12 gene mutations in women with uterine myoma. Genetika. 2013;49:1426–31. [PubMed] [Google Scholar]
- 37.Perot G, Croce S, Ribeiro A, Lagarde P, Velasco V, Neuville A, et al. MED12 alterations in both human benign and malignant uterine soft tissue tumors. PloS one. 2012;7:e40015. doi: 10.1371/journal.pone.0040015. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.Wang H, Ye J, Qian H, Zhou R, Jiang J, Ye L. High-resolution melting analysis of MED12 mutations in uterine leiomyomas in Chinese patients. Genetic testing and molecular biomarkers. 2015;19:162–6. doi: 10.1089/gtmb.2014.0273. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39.Mäkinen N, Heinonen HR, Moore S, Tomlinson IP, van der Spuy ZM, Aaltonen LA. MED12 exon 2 mutations are common in uterine leiomyomas from South African patients. Oncotarget. 2011;2:966–9. doi: 10.18632/oncotarget.370. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.Croce S, Chibon F. MED12 and uterine smooth muscle oncogenesis: State of the art and perspectives. Eur J of Can. 2015;51:1603–10. doi: 10.1016/j.ejca.2015.04.023. [DOI] [PubMed] [Google Scholar]
- 41.Taatjes DJ. The human Mediator complex: a versatile genome-wide regulator of transcription. Trends Biochem Sci. 2010;35:315–22. doi: 10.1016/j.tibs.2010.02.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 42.Taatjes DJ, Naar AM, Andel F, III, Nogales E, Tijan R. Structure, function, and activator-induced conformations of the CRSP coactivator. Science. 2002;295:1058–62. doi: 10.1126/science.1065249. [DOI] [PubMed] [Google Scholar]
- 43.Mittler G, Kremmer E, Timmers HT, Meisterernst M. Novel critical role of a human Mediator complex for basal RNA polymerase II transcription. EMBO Rep. 2001;2:808–13. doi: 10.1093/embo-reports/kve186. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44.Akoulitchev S, Chuikov S, Reinberg D. TFIIH is negatively regulated by cdk8-containing mediator complexes. Nature. 2000;407:102–6. doi: 10.1038/35024111. [DOI] [PubMed] [Google Scholar]
- 45.Knuesel MT, Meyer KD, Bernecky C, Taatjes DJ. The human CDK8 subcomplex is a molecular switch that controls Mediator coactivator function. Genes Dev. 2009;23:439–51. doi: 10.1101/gad.1767009. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 46.van de Peppel J, Kettelarij N, van BH, Kockelkorn TT, van LD, Holstege FC. Mediator expression profiling epistasis reveals a signal transduction pathway with antagonistic submodules and highly specific downstream targets. Mol Cell. 2005;19:511–22. doi: 10.1016/j.molcel.2005.06.033. [DOI] [PubMed] [Google Scholar]
- 47.Donner AJ, Ebmeier CC, Taatjes DJ, Espinosa JM. CDK8 is a positive regulator of transcriptional elongation with the serum response network. Nat Struct Mol Biol. 2010;17:194–201. doi: 10.1038/nsmb.1752. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 48.Firestein R, Bass AJ, Kim SY, Dunn IF, Silver SJ, Guney I, et al. CDK8 is a colorectal cancer oncogene that regulates beta-catenin activity. Nature. 2008;455:547–51. doi: 10.1038/nature07179. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 49.Morris EJ, Ji JY, Yang F, Stefano L, Herr A, Moon NS, et al. E2F1 represses beta-catenin transcription and is antagonized by both pRB and CDK8. Nature. 2008;455:552–6. doi: 10.1038/nature07310. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 50.Turunen M, Spaeth JM, Keskitalo S, Park MJ, Kivioja T, Clark AD, et al. Uterine leiomyoma-linked MED12 mutations disrupt mediator-associated CDK activity. Cell Rep. 2014;7:654–60. doi: 10.1016/j.celrep.2014.03.047. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 51.Mehine M, Mäkinen N, Heinonen HR, Aaltonen LA, Vahteristo P. Genomics of uterine leiomyomas: insights from high-throughput sequencing. Fertil Steril. 2014;102(3):621–29. doi: 10.1016/j.fertnstert.2014.06.050. [DOI] [PubMed] [Google Scholar]
- 52.Mehine M, Heinonen HR, Sarvilinna N, Pitkänen E, Mäkinen N, Katainen R, et al. Clonally related uterine leiomyomas are common and display branched tumor evolution. Hum Mol Genet. 2015;24(15):4407–16. doi: 10.1093/hmg/ddv177. [DOI] [PubMed] [Google Scholar]
- 53.Holzmann C, Markowski DN, Koczan D, Küpker W, Helmke BM, Bullerdiek J. Cytogenetically normal uterine leiomyomas without MED12-mutations - a source to identify unknown mechanisms of the development of uterine smooth muscle tumors. Mol Cytogenet. 2014;7(1):88. doi: 10.1186/s13039-014-0088-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 54.Holzmann C, Markowski DN, Bartnitzke S, Koczan D, Helmke BM, Bullerdiek J. A rare coincidence of different types of driver mutations among uterine leiomyomas (UL) Mol Cytogenet. 2015;8:76. doi: 10.1186/s13039-015-0177-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 55.Holzmann C, Markowski DN, VON Leffern I, Löning T, Bullerdiek J. Patterns of chromosomal abnormalities that can improve diagnosis of uterine smooth muscle tumors. Anticancer Res. 2015;35(12):6445–56. [PubMed] [Google Scholar]
- 56.Harris PA, Taylor R, Thielke R, Payne J, Gonzalez N, Conde JG. Research electronic data capture (REDCap)--a metadata-driven methodology and workflow process for providing translational research informatics support. Journal of biomedical informatics. 2009;42:377–81. doi: 10.1016/j.jbi.2008.08.010. [DOI] [PMC free article] [PubMed] [Google Scholar]