Mitochondrial displacement loop alterations are associated with endometriosis

Mitochondrial displacement loop alterations are associated with endometriosis Suresh Govatati, M.Sc.,a Mamata Deenadayal, M.D.,b Sisinthy Shivaji, Ph.D.,c and Manjula Bhanoori, Ph.D.a a Department of Biochemistry, Osmania University, Hyderabad; b Infertility Institute and Research Centre, Secundrabad; and c Centre for Cellular and Molecular Biology (CCMB), Hyderabad, India

Objective: To evaluate the association of mitochondrial displacement (D-) loop alterations with endometriosis in south Indian women. Design: Case-control study. Setting: Biochemistry and molecular biology laboratories. Patient(s): Women with (n ¼ 152) and without (n ¼ 150) endometriosis of south Indian origin. Intervention(s): All women had a transvaginal ultrasound scan at screening followed by a laparoscopy to confirm the diagnosis (revised American Fertility Society stage III ¼ 54; stage IV ¼ 98). Main Outcome Measure(s): Genotyping of entire D-loop (1,124 bp) was carried out on genomic DNA of blood from cases and controls by polymerase chain reaction sequencing analysis. Result(s): Twelve novel mutations and 187 reported polymorphisms were identified in the D-loop region of cases and/or controls. The A189G, 310 C insertion, T16189C polymorphisms, and 189G/310TC/16189C haplotype have significantly higher frequency in cases compared with controls. Conclusion(s): Mitochondrial D-loop alterations may constitute an inheritable risk factor for endometriosis. The analysis of D-loop alterations may help to identify patients at high risk Use your smartphone for disease outcome. (Fertil SterilÒ 2013;99:1980–6. Ó2013 by American Society for Reproducto scan this QR code tive Medicine.) and connect to the Key Words: Endometriosis, mitochondria, D-loop, polymorphism Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/govatatis-mitochondrial-d-loop-endometriosis/

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ndometriosis (MIM 131200) is a complex gynecological disorder characterized by the presence of endometrial glands and stroma within extrauterine sites, most commonly the ovaries and peritoneum. It affects approximately 5%–10% of women of reproductive age and up to 50% of women with infertility (1). Although symptoms vary, they commonly include severe pelvic pain, severe dysmenorrhea, and subfertility.

The causes of endometriosis remain uncertain, despite over 50 years of hypothesis-driven research. Previously we demonstrated the correlation between various candidate genes and endometriosis risk in Indian women (2–8). Recent investigations have shown the potential involvement of reactive oxygen species (ROS) in the pathogenesis of endometriosis (9–11). Mitochondria are a major source for ROS generation, and mitochondrial

Received November 12, 2012; revised January 28, 2013; accepted February 13, 2013; published online March 13, 2013. S.G. has nothing to disclose. M.D. has nothing to disclose. S.S. has nothing to disclose. M.B. has nothing to disclose. This study was supported in part by grants from the Department of Science and Technology (DST), India (Lr No: SR/FT/LS-188/2009), and OU-DST PURSE to M.B. Reprint requests: Manjula Bhanoori, Ph.D., Assistant Professor, Department of Biochemistry, Osmania University, Hyderabad 500 007, India (E-mail: [email protected]). Fertility and Sterility® Vol. 99, No. 7, June 2013 0015-0282/$36.00 Copyright ©2013 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2013.02.021 1980

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DNA (mtDNA) alterations have been found in a variety of human diseases (12). Mitochondria are subcellular organelles responsible for the production of cellular ATP through the process of oxidative phosphorylation (OXPHOS) (12). In addition to OXPHOS, mitochondria are important regulators of aging, ROS generation, and apoptosis (12). Hence mitochondria may serve as the switching point between cell death and abnormal cell growth. The human mtDNA is a closed circular duplex molecule (16.569 kb) that contains 37 genes coding for two ribosomal RNAs, 22 tRNAs, and 13 polypeptides (13). It presents high copy levels (103–104 copies per cell) in virtually all cells, and the vast majority of copies are identical at birth (14). MtDNA is more vulnerable to oxidative VOL. 99 NO. 7 / JUNE 2013

Fertility and Sterility® damage and acquires mutations at a higher rate than nuclear DNA. This is due to the absence of protecting histones, higher levels of ROS, and the lack of an efficient DNA repair system in mitochondria (15). The displacement loop (D-loop) is the only noncoding region (nucleotide position [np] 16024–576 ¼ 1,124 bp) of the mitochondrial genome and is known to accumulate mutations at a higher frequency than other regions (16). It is a hot spot for mtDNA alterations and contains two hypervariable regions (HVR1: np 16024–16383; and HVR2: np 57–333). The D-loop contains crucial elements for replication (heavy chain) and transcription (heavy and light chains) of mtDNA (17). Hence, sequence alterations of the D-loop may contribute to altered replication and/or transcription of mitochondrial genes, which may affect the overall mitochondrial function and cellular ROS generation. Accumulation of D-loop alterations has been reported in several complex human diseases (16, 18–20), but they have not yet been systematically characterized in endometriosis. In this study we report for the first time a comprehensive study of D-loop alterations in endometriosis.

MATERIALS AND METHODS

Genotyping of the Entire D-Loop Genomic DNA was extracted from 1 mL of EDTA anticoagulated whole blood by the salting-out method (23). The entire D-loop region mutations were analyzed by polymerase chain reaction (PCR) sequencing analysis as per the protocols described elsewhere (8). Two pairs of overlapping primers (Supplemental Table 1) were used to amplify the entire 1,122 bp D-loop region of mtDNA. The generated DNA fragments vary in size from 809 to 963 bp, with an average of 886 bp. The amplified fragments totaled 1,772 bp, 63.43% more than the whole D-loop because of the overlapping regions.

Mutational Analysis of Entire D-Loop The individual mtDNA sequences were compared against the Revised Cambridge Reference Sequence (rCRS) (24) using Auto Assembler (ver. 2.1; Applied Biosystems). Sequences were aligned using CLUSTAL X, and mutations were noted by using MEGA software version 3.1. Independent sequencing readings were performed by two different individuals (S.G. and M.B.). Sequence variations found in both cases and controls were checked against the Mitomap database. Those not recorded in the database were categorized as novel mutations, and those that appeared in the database were reported as polymorphisms.

Sample Collection and Diagnosis A total of 152 premenopausal unrelated south Indian women (Dravidian linguistic group) with moderate-severe (III–IV) endometriosis staged using the revised American Fertility Society (rAFS) classification system (21) were recruited at the Infertility Institute and Research Centre, Hyderabad, India. All women had a transvaginal ultrasound scan (TVS) at screening followed by a laparoscopy to confirm the diagnosis (rAFS stage III ¼ 54; stage IV ¼ 98). All the patients had different forms of endometriosis such as peritoneal lesions, adhesions, and endometrioma. Women with other ovarian cysts, adenomyosis, ovarian cancer, fibroids, and stage I and II endometriosis were excluded from the study. The aim was to focus on patients with more severe endometriosis (stages III and IV) because the more severe forms include ovarian cystic disease, which almost certainly has a different etiology from peritoneal forms, and the diagnosis is usually unequivocal, which is not the case for stages I and II. Their mean age  SD was 26.3  5.6 (range, 20–40) years. All patients complained of dysmenorrhea (mild, 44%; moderate, 30%; severe, 26%), and 76% had dyspareunia. Most women (97.1%) were infertile (primary, 82%; secondary, 18%). One hundred fifty women were recruited from the same clinic population and had an equal opportunity to be identified as cases, thereby meeting the criteria for appropriate controls set by Zondervan et al. (22). The controls consisted of women with no evidence of endometriosis on TVS and laparoscopy. Their mean age  SD was 27.6  4.93 (range, 22–40) years. Seventy-three percent had primary and 27% had secondary infertility. Among the controls, 27% complained of mild dysmenorrhea and 15% complained of dyspareunia. Written informed consent was obtained from all participants. The Institutional Review Board of the Centre for Cellular and Molecular Biology, Hyderabad, approved the study. VOL. 99 NO. 7 / JUNE 2013

Statistical Analysis Statistical analysis was performed using the SPSS statistical package (ver. 11.0). The allele ratios and genotype distributions of cases and controls were analyzed using Fisher's exact test. The odds ratio and 95% confidence interval (CI) values were calculated using the online Vassar Stats Calculator (http://www.faculty.vassar.edu/lowry/VassarStats.html). Haplotype frequencies for multiple loci and the standardized disequilibrium coefficient (D') for pair-wise linkage disequilibrium (LD) were assessed by Haploview software (25). P<.05 was considered statistically significant. The Bonferroni correction was used to adjust the significance level of a statistical test to protect against type I errors when multiple comparisons were being made.

RESULTS D-Loop Alterations in Endometriosis Genotyping of the entire mitochondrial D-loop region was successfully completed in all patients (n ¼ 152) and controls (n ¼ 150). Patients with endometriosis (1,199/152 ¼ 7.88) showed comparatively higher single nucleotide polymorphism (SNP) frequency compared with the numbers of SNPs identified in each individual than controls (804/150 ¼ 5.36). In this study, we identified 12 novel mutations in the D-loop region of patients with endometriosis (Fig. 1; Supplemental Table 2). Among them, two were nucleotide deletions, five were nucleotide insertions, and five were base substitutions. In addition, we identified 187 reported polymorphisms in the D-loop region of cases and/or controls (Table 1; Supplemental Table 3). Most of them were single base substitutions (Y or R transitions). Overall, among the identified 187 reported polymorphisms, 167 were base substitutions, 12 were nucleotide deletions, seven were nucleotide insertions, 1981

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FIGURE 1

Novel mutations observed in the mitochondrial D-loop region of patients with endometriosis. (A) A16T transversion; (B) 56A deletion; T58A transversion; 65T insertion; (C) 105–110CGGAGC deletion; (D) C113T transition; (E) 149T insertion (152T deletion, a reported mutation also shown); (F) C394T transition; (G) 401CAAATTTT insertion; (H) 524CA insertion; (I) 16225C insertion (16223C deletion, a reported mutation also shown); (J) T16351C transition. Govatati. Mitochondrial D-loop and endometriosis. Fertil Steril 2013.

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TABLE 1 Mitochondrial D-loop polymorphisms with >5% minor allele frequency observed in patients with endometriosis and/or controls. Serial number

Nucleotide position

rCRS

Base change

IUPAC code

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

A73G A93G T146C T152C A189G T195C T195A T246C A263G 310C ins 316C ins T489C CA522 del C525 del A16051G G16129A T16172C T16189C

A A T T A T T T A T C T CA C A G T T

G G C C G C A C G TC CC C – – G A C C

R R Y Y R Y W Y R

19 20 21 22 23 24 25 26 27

C16223T T16231C C16266T C16278T T16311C A16318T G16319A T16362C T16519C

C T C C T A G T T

T C T T C T A C C

Frequency Cases

Controls

P valuea

c2

R R Y Y

92 11 24 22 49 11 17 8 96 51 91 43 21 12 13 15 9 41

81 9 17 28 33 7 13 3 81 34 79 37 18 13 9 7 11 17

.251 .665 .258 .3269 .0455 .3454 .4645 .1301 .1061 .0354 .2071 .4755 .6382 .8080 .3935 .0819 .6213 .0005

1.314 0.187 1.278 0.961 4.0 0.89 0.535 2.291 2.61 4.424 1.591 0.509 0.221 0.059 0.728 3.025 0.244 11.902

Y Y Y Y Y W R Y Y

57 9 11 10 17 8 14 17 78

48 5 8 7 11 3 16 12 72

.3156 .2848 .4957 .4708 .2486 .1301 .6722 .3476 .5644

1.007 1.144 0.464 0.52 1.331 2.291 0.179 0.882 0.332

Y

Association

Leukemia

Type 2 diabetes; cardiomyopathy Endometrial carcinoma

Note: IUPAC ¼ International Union of Pure and Applied Chemistry. a Fisher's exact test (2  2 table at 1 degree of freedom); P<.05. Govatati. Mitochondrial D-loop and endometriosis. Fertil Steril 2013.

and one was a heteroplasmic mutation. Polymorphisms were predominantly located in HVR1 (64.7%) compared with in HVR2 (22.5%) of the D-loop region. Twenty-seven of the 187 reported polymorphisms have >5% minor allele frequency in either patients or controls (Table 1). These were subjected to further statistical analysis. Three of them, A189G (P¼ .0455; Supplemental Fig. 1), 310 C insertion (P¼ .0354; Supplemental Fig. 2), and T16189C (P¼ .0005; Supplemental Fig. 3), have a significantly higher frequency in patients with endometriosis compared with in controls. Genotype frequencies of significant D-loop polymorphisms were further analyzed based on the stage (rAFS III and IV) and size of the largest endometrioma present (Supplemental Table 4). For the T16189C polymorphism, statistically significant differences in the frequencies of the T and C alleles were observed between controls and all patient groups. A higher frequency of 310 C insertion was found in patients with an endometrioma 4–5 cm (P¼ .01658) and rAFS stage IV (P¼ .01363) compared with in controls. However, for the A189G polymorphism, statistically significant differences in the frequencies of the A and G alleles were observed only between controls and patients with an endometrioma >6 cm (P¼ .03621).

Haplotype Analysis To analyze the combined effect of significant D-loop SNPs in the pathogenesis of endometriosis, the haplotype frequencies VOL. 99 NO. 7 / JUNE 2013

for significant loci and the standardized disequilibrium coefficient (D') for pair-wise LD were estimated (Table 2; Fig. 2). Our results showed a different pattern of LD between patients and controls. Particularly, the 189A/G and 16189T/C loci showed moderately strong LD in patients (D' ¼ 0.24) compared with in controls (D' ¼ 0.02). Furthermore, our data indicate 189A/310T/16189T as the most common haplotype in south Indian women. The relative risk of each haplotype was calculated by using this haplotype as a reference. Bonferroni correction was used to adjust the significance level of a statistical test to protect against type I errors. Since we have eight haplotypes, the Bonferroni correction should be 0.05/8 ¼ 0.00625. Therefore, P<.00625 was considered statistically significant. Our results indicate that the 189G/310TC/16189C (P¼ .00055) haplotype significantly increases the risk of developing endometriosis, while the remaining haplotypes were not indicative for the disease risk.

DISCUSSION Endometriosis is a benign but invasive gynecological disease. Clinical observations and in vitro experiments revealed the invasive and proliferative nature of endometriotic cells (26). ROS play a pivotal role in cellular proliferation and neoplastic growth (27–29). It has been suggested that ROS may increase growth and adhesion of endometrial cells in the peritoneal 1983

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TABLE 2 Haplotype frequencies of significant D-loop polymorphisms observed in patients with endometriosis and controls. Haplotypes 189

Haplotype frequency 310

16189

Cases

Controls

P valuea

c2

Odds ratio

95% CI

T T TC TC T T TC TC

T C T C T C T C

57 16 25 5 20 8 9 12

78 11 26 2 24 3 5 1

.1043 .4048 .1287 .7072 .0502 .1136 .00055

Reference 2.638 0.694 2.308 0.141 3.832 2.503 11.953

0.5046 0.7601 0.3076 0.8771 0.2933 0.4114 0.1355

0.2205–1.1548 0.398–1.4519 0.0668–1.4156 0.442–1.7405 0.0855–1.0058 0.1364–1.2411 0.0435–0.4224

A A A A G G G G

Note: P<.00625 (after Bonferroni correction). a Fisher's exact test (2  2 table at 1 degree of freedom). Govatati. Mitochondrial D-loop and endometriosis. Fertil Steril 2013.

cavity, promoting endometriosis and infertility (30, 31). The mitochondrial D-loop is a hot spot for mtDNA alterations and is important for the regulation of mitochondrial genome replication and expression (17). Hence, D-loop alterations may lead to alterations in electron transport chain (ETC) and cellular ROS generation. This directed our attention to this mtDNA region. Recently, we reported somatic/noninheritable mtDNA alterations in endometriosis (8). However, the strong familial tendency of the disease suggests a possible inheritable genetic susceptibility. The inheritance pattern of the mitochondrial genome (maternal) leads to the gradual accumulation of mutations in successive generations. Hence, the altered mitochondrial alleles may act as inheritable predisposing factors for several diseases (12). An association among three common germ line/inheritable mtDNA polymorphisms (two from complex-I: A10398G and G13708A; and one from the D-loop: T16189C) and endometriosis has recently been reported (32). However, none of the investigators have reported entire D-loop germ line alterations in endometriosis. In this study, for the first time, we determined

FIGURE 2

LD analyses of significant D-loop SNPs in cases and controls are shown separately. Haploview plots are presented along the SNPs studied. The pair-wise LD values (D' ¼ 0–100) of all SNPs are given in each diamond. A value of 100 represents the maximum possible LD. Govatati. Mitochondrial D-loop and endometriosis. Fertil Steril 2013.

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the frequencies of entire D-loop germ line alterations in endometriosis. SNPs appear to be common in this south Indian population, with an average of five to eight for each individual in reference to rCRS. The actual number of SNPs may be less if the reference sequence was of Indian origin. When compared with controls, frequent SNPs in patients with endometriosis provide the first evidence that a high SNP frequency in the Dloop seems to predispose to endometriosis. Furthermore, our results showed a significant association among D-loop SNPs at np 189 (A/G), 310 (C insertion), and 16189 (T/C) and endometriosis risk. Interestingly, among the three identified significant SNPs, two (310 and 16189) were located in the microsatellite loci of mtDNA. The 310 C insertion, the most common D-loop variant/ microsatellite instability (MSI), has been associated with various multifactorial disorders (19, 33). The np 310 was located within a homopolymeric C-stretch between np 303 and 315 interrupted by thymine (Supplemental Fig. 2). This poly-C stretch is situated in the HVR II (np 57–333) and was reported to be a mutational hotspot (34). Furthermore, it is the replication primer binding site and located in the heavystrand conserved sequence block II (CSB II), which contributes to the formation of a persistent RNA-DNA hybrid to initiate the mtDNA replication (35). The RNA-DNA hybrid's formation is dependent on this GC-rich element. Interestingly, efficient hybrid formation is also influenced by sequences 5' to the hybrid, including the CSB II element (36). In addition, mtDNA transcription is critically dependent on the exact CSB II sequence that is required to generate the RNA primers used in the initiation of heavy-strand DNA synthesis (37). Premature transcription termination and drastic reduction in transcription occurs if particular MSI arise in np 282–300 and 304–300 of the mtDNA sequences, respectively, whereas complete termination occurs in the 289–319 mutants (38). Thus severe alterations in this repeat could lead to functional impairment of mitochondria and may result in a protumorigenic phenotype of the carrier. The T16189C variant, a strong hotspot of mitochondrial MSI with a (C)7–14 pattern of variation (37), has been associated with several multifactorial disorders (20, 39). The T/C transition at np 16189 generates an uninterrupted poly-C tract (np 16180–16195) in the D-loop region (Supplemental VOL. 99 NO. 7 / JUNE 2013

Fertility and Sterility® Fig. 3). This SNP may also lead to heteroplasmic length variation of the poly-C tract (>10 cytosines) in different mtDNA molecules of a single person (40). Mitochondrial single-strand DNA-binding protein bound less efficiently to uninterrupted poly-C variant when compared with the interrupted poly-C (20). Furthermore, nucleotides 16184–16193 are located on the 3'-end of a termination-associated sequence and at the 7S DNA binding site, which are thought to be involved in the regulation of mtDNA synthesis (41). Hence, the uninterrupted poly-C variant may reduce mitochondrial replication and mtDNA content. Reduced mtDNA content could affect the efficiency of the ETC, lower the ATP:ADP ratio, and enhance the ROS generation (12, 15). Elevated ROS generation and lowered ATP:ADP ratio could contribute to the onset of several multifactorial diseases (29, 33). In our study, the variant with the uninterrupted poly-C showed significantly higher frequencies in endometriosis cases compared with in controls (P¼ .0005). Moreover, the 189G/310TC/16189C (P¼ .00055) haplotype may be associated with an increased risk for endometriosis. Interestingly, the 189G/310TC/16189C haplotype has mutated alleles at all three loci. As per our results, it is a rare haplotype in south Indian women (Table 2). However, its relative frequency in the cases was significantly higher than that in the controls. It indicates the significance of this haplotype in the pathogenesis of endometriosis. Furthermore, these three mutated alleles may alter the replication and/or transcription of the mitochondrial genome, which may lead to enhanced ROS production. Elevated ROS generation has been reported in several human diseases (12, 15). Estrogeninducible diseases like endometriosis are more susceptible to the effects of mitochondrial dysfunction because the normal metabolism of E2 through redox cycling intermediates may also generate the local ROS and oxidative damage that facilitates neoplastic transformation (42). ROS has been shown to be involved in increased growth and adhesion of endometrial cells in the peritoneal cavity (30, 31, 43). Higher endogenous oxidative stress and alterations in ROS detoxification pathways have been reported in endometriotic cells (11). All these observations strongly correlate the mitochondrial dysfunction with endometriosis risk. In conclusion, mitochondrial D-loop alterations may constitute an inheritable risk factor for endometriosis. The analysis of genetic alterations in the D-loop might help to identify patients at high risk for disease outcome. To the best of our knowledge, this is the first report demonstrating the correlation between entire D-loop alterations and endometriosis risk in humans. Future experiments with a large sample size and functional evaluation of identified SNPs are needed to validate our findings in this pilot study. Acknowledgments: The authors thank the patients who participated in the present study. Suresh Govatati would like to thank the Council of Scientific and Industrial Research, India for JRF (NET) and SRF (NET).

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SUPPLEMENTAL FIGURE 1

Genotyping of the mitochondrial D-loop A189G polymorphism by sequence analysis of the PCR amplified product using a forward primer. An additional polymorphism at nucleotide position G185A is also shown. Govatati. Mitochondrial D-loop and endometriosis. Fertil Steril 2013.

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SUPPLEMENTAL FIGURE 2

Genotyping of the mitochondrial D-loop 310 C insertion polymorphism by sequence analysis of the PCR-amplified product using a forward primer. Govatati. Mitochondrial D-loop and endometriosis. Fertil Steril 2013.

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SUPPLEMENTAL FIGURE 3

Genotyping of the mitochondrial D-loop T16189C polymorphism by sequence analysis of the PCR-amplified product using a forward primer. Govatati. Mitochondrial D-loop and endometriosis. Fertil Steril 2013.

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SUPPLEMENTAL TABLE 1 Primers used in this study for entire D-loop sequencing. Serial number

Primer set

Primer sequence 5'/3'

Nucleotide position

Overlap (bp)

Amplicon size (bp)

Tana ( C)

1F 1R 2F 2R

TCATTGGACAAGTAGCATCC GAGTGGTTAATAGGGTGATAG CACCATCCTCCGTGAAATCA AGGCTAAGCGTTTTGAGCTG

1579231 16401794

–

809

58

199

963

58

1 2 a

Annealing temperature.

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SUPPLEMENTAL TABLE 2 Novel mutations detected in mitochondrial D-loop region of patients with endometriosis. Serial number

Nucleotide position

1 2 3 4 5 6 7 8 9 10 11 12

A16T A56 del T58A 65T ins 105-110 del C113T 149T ins C394T 401 ins 524 ins 16225C ins T16351C

rCRS

Base change

IUPAC code

Frequency

A A T – CGGAGC C – C – – – T

T – A T – T T T CAAATTTT CA C C

W

1 1 1 1 2 1 1 1 3 4 1 3

W Y Y

Y

Note: del ¼ deletion; ins ¼ insertion; IUPAC ¼ International Union of Pure and Applied Chemistry. Govatati. Mitochondrial D-loop and endometriosis. Fertil Steril 2013.

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ORIGINAL ARTICLE: ENDOMETRIOSIS

SUPPLEMENTAL TABLE 3 Mitochondrial D-loop polymorphisms with <5% minor allele frequency observed in patients with endometriosis and/or controls. Frequency Serial number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Nucleotide position

rCRS

Base change

IUPAC code

Cases

Controls

T89C C114T C114 del 150T ins C150T C151 del C151T 152C ins T152 del G185A 193T ins C194T T196 del T199C A200G G203A T204C G207A T217C G228A A234G A249 del T250C A270G C271T T279C T282C C285T C295T 309C ins C324T A373G C431T C447A C459T C462T T480C T482C A508G C511T 525C ins C568T C569T A574C A16037G A16038G G16048A A16059G A16066G C16069T C16071T T16075C T16086C T16092C T16093C T16093T/C C16111T T16126C G16129C C16134T T16136C T16144C G16145A T16154C C16150T

T C C – C C C – T G – C T T A G T G T G A A T A C T T C C – C A C C C C T T A C – C C A A A G A A C C T T T T T C T G C T T G T C

C T – T T – T C – A T T – C G A C A C A G – C G T C C T T C T G T A T T C C G T C T T C G G A G G T T C C C C T/C T C C T C C A C T

Y Y

1 2 2 4 2 3 1 3 5 1 1 4 1 4 2 1 5 3 3 3 5 1 1 1 1 1 3 1 3 1 1 2 1 3 3 3 1 3 3 1 4 1 2 4 1 1 1 1 1 3 1 1 3 5 4 2 3 6 3 2 1 1 4 1 1

3 0 0 1 0 1 1 0 2 0 2 0 0 5 1 1 2 4 2 0 0 1 2 0 0 0 0 2 0 0 0 0 0 0 1 0 1 1 0 0 0 1 0 0 0 0 1 0 0 0 0 0 1 3 3 0 0 1 0 0 0 1 5 0 0

Y Y R Y Y R R Y R Y R R Y R Y Y Y Y Y Y R Y M Y Y Y Y R Y Y Y M R R R R R Y Y Y Y Y Y Y Y S Y Y Y R Y Y

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SUPPLEMENTAL TABLE 3 Continued. Frequency Serial number 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

Nucleotide position

rCRS

G16153A T16154C A16166G A16166 del C16167T C16168T A16170T T16171C C16176T T16178C C16179T A16182C A16183C A16183 del C16185T C16186T C16187T C16188T T16189 del C16192T C16193T A16194C T16195C A16206C A16207G T16209C A16215G T16217C A16220G C16221T C16222T C16223 del T16224C A16227G A16230G C16234T C16239T A16240C C16242T A16243C C16245T A16247G C16248T T16249C A16254G G16255A C16256T C16257A C16259T C16260T C16261T C16262T T16263C C16264T A16265C C16270T G16274A C16286T C16287T A16289G C16290T C16291T C16292T A16293G C16294T

G T A A C C A T C T C A A A C C C C T C C A T A A T A T A C C C T A A C C A C A C A C T A G C C C C C C T C A C G C C A C C C A C

Base change A C G – T T T C T C T C C – T T T T – T T C C C G C G C G T T – C G G T T C T C T G T C G A T A T T T T C T C T A T T G T T T G T

IUPAC code

Cases

Controls

R Y R

2 2 4 1 1 2 1 1 3 1 4 4 5 3 1 1 1 5 1 1 1 1 1 1 2 5 1 3 1 1 1 1 1 2 1 4 4 1 1 1 2 1 3 3 1 1 2 2 4 2 6 1 1 1 1 2 6 1 1 4 1 1 2 1 1

1 2 0 1 0 0 0 0 0 1 1 0 3 4 0 0 1 0 0 0 0 1 1 2 0 3 3 1 0 0 0 1 0 0 0 1 5 0 0 1 2 0 1 0 0 1 0 0 1 0 3 0 0 1 1 0 3 0 1 5 0 0 1 0 2

Y Y W Y Y Y Y M M Y Y Y Y Y Y M Y M R Y R Y R Y Y Y R R Y Y M Y M Y R Y Y R R Y M Y Y Y Y Y Y M Y R Y Y R Y Y Y R Y

Govatati. Mitochondrial D-loop and endometriosis. Fertil Steril 2013.

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ORIGINAL ARTICLE: ENDOMETRIOSIS

SUPPLEMENTAL TABLE 3 Continued. Frequency Serial number 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160

Nucleotide position

rCRS

C16295T C16296T T16298C A16300G C16301T T16304C A16309G T16325C C16327T A16343T C16344T T16352C C16353T C16354T C16355 del T16356C T16357C T16359C C16360T T16362A C16365T T16368C A16374C G16390A G16391A A16399G A16463G A16497G A16524G C16527T

C C T A C T A T C A C T C C C T T T C T C T A G G A A A A C

Base change T T C G T C G C T T T C T T – C C C T A T C C A A G G G G T

IUPAC code

Cases

Controls

Y Y Y R Y Y R Y Y W Y Y Y Y

1 2 1 1 2 6 3 1 2 2 1 3 1 1 4 2 1 2 1 3 3 1 1 2 1 1 1 1 3 3

1 0 0 1 0 0 1 1 0 1 0 1 5 0 0 1 2 0 1 0 1 0 0 0 1 2 0 0 2 0

Y Y Y Y W Y Y M R R R R R R Y

Note: IUPAC ¼ International Union of Pure and Applied Chemistry. Govatati. Mitochondrial D-loop and endometriosis. Fertil Steril 2013.

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SUPPLEMENTAL TABLE 4 Genotype frequencies of significant D-loop polymorphisms in patients with endometriosis based on different clinical parameters. Endometriotic cyst size of casesa

rAFS stage of cases Polymorphism

III (n [ 54)

IV (n [ 98)

<3 (n [ 9)

4–5 (n [ 47)

>6 (n [ 96)

117 (78) 33 (22)

37 (68.5) 17 (31.5) .12912

66 (67.3) 32 (32.7) .08963

7 (77.8) 2 (22.2) .97477

34 (72.3) 13 (27.7) .35096

62 (64.6) 34 (35.4) .03621

116(77.3) 34 (22.7)

41 (75.9) 13 (24.1) .08963

60 (61.2) 38 (38.8) .01363

7 (77.8) 2 (22.2) .93332

29 (61.7) 18 (38.3) .01658

65 (67.7) 31 (32.3) .12846

133 (88.7) 17 (11.3)

40 (74.1) 14 (25.9) .00798

71 (72.4) 27 (27.6) .00359

6 (66.7) 3 (33.3) .00019

33 (70.2) 14 (29.8) .00121

72 (75) 24 (25) .01196

Controls (n [ 150)

A189G A G Pb 310C ins T TC Pb T16189C T C Pb

Note: Data are n (%). a In centimeters. b Fisher's exact test (2  2 table at 1 degree of freedom); P<.05. Govatati. Mitochondrial D-loop and endometriosis. Fertil Steril 2013.

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