Mitochondrial DNA deletions associated with endometriosis

Abstract

Abnormal mitochondrial DNA (mtDNA) molecules are provided that have specific large deletions and are associated with endometriosis. The abnormal or mutated mtDNA may contain the parent nucleic acid (i.e., the major subunit), particularly when recircularized, with adjacent nucleotides fused after the deletion to form a junction site. Alternatively, the mtDNA may contain the deleted strand (i.e., the minor subunit), particularly when recircularized to create a junction site. Furthermore, fusion transcripts resulting from such mutated mtDNA and their predicted protein products are provided, and such transcripts and proteins are also associated with endometriosis. Hybridization probes and amplification primers, as well as kits containing them, are provided for detecting, diagnosing, or monitoring endometriosis.

Description

Detailed Description of the Invention

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under the Paris Convention to U.S. Application No. 62 / 784,403, filed December 22, 2018, and U.S. Application No. 62 / 931,173, filed November 5, 2019. The entire contents of such prior applications are incorporated herein by reference.

[0002] [Explanation regarding sequence listing] The sequence listing associated with this application has been submitted contemporaneously herewith in ASCII format and is incorporated herein by reference. The text file containing the sequence listing is titled "Sequence_listing.txt", was created on December 17, 2019, and is approximately 119 kilobytes in size.

[0003] [Technical field to which the description pertains] SUMMARY OF THE DISCLOSURE The present description generally relates to novel biomarkers and methods for detecting / diagnosing and / or monitoring endometriosis. The description also relates to unique analytes and / or reagents useful in the subject methods.

[0004] 〔background〕 Endometriosis is a burdensome disease affecting up to 5–10% of women of reproductive age and a frequent cause of infertility [1–7,58]. The disease is characterized by the presence of endometrial tissue (epithelial cells and stromal) proliferating outside the uterus. Such ectopic endometrial tissue can be found in the pelvic peritoneum and fallopian tubes, ovaries, bowel and bladder, as well as in more distal body sites [8–11]. Women with endometriosis frequently suffer from debilitating symptoms, including nonmenstrual pelvic pain, painful menstrual cramps, pain during intercourse, fatigue, and infertility

[12] , which can lead to a significant decrease in quality of life

[13] . Given its high prevalence and significant morbidity, endometriosis results in very significant economic losses worldwide, estimated at tens of billions of euros annually

[14] .

[0005] Unfortunately, diagnosing endometriosis is often a lengthy process, resulting in delayed treatment. The current "gold standard" for diagnosing endometriosis is laparoscopic surgery followed by histopathological confirmation of tissue samples [5,15]. Making a timely diagnosis is further complicated by delayed reporting

[16] and misinterpretation of symptoms

[17] , and may be further delayed if patients are unwilling to undergo expensive and invasive laparoscopic procedures. In fact, delays in diagnosing endometriosis can exceed 10 years

[16] . These delays may result in the majority of women developing moderate to severe symptoms by the time a definitive diagnosis is made, resulting in increased morbidity, treatment costs, and reduced quality of life

[14] . Thus, there is a need for a reliable, noninvasive test that can facilitate early detection of endometriosis and provide actionable real-time results. However, no noninvasive methods for detecting endometriosis are currently available.

[0006] Molecular biomarkers are widely used as tools to measure, detect, and predict human disease [18–24]. However, the search for endometriosis-specific biomarkers has proven challenging

[25] . Some major challenges include nonstandardized specimen collection, analytical methods, and data interpretation as well as lack of biomarker specificity

[17] , although recent efforts have been made to harmonize methods of biological specimen collection and storage with reporting of endometriosis data, including the World Endometriosis Research Foundation (WERF) EPHect protocol

[26] . Although various candidate biomarkers from blood, tissue, and urine have been reported, none have been successfully translated into clinical use. Many of these candidates have inherent limitations in sample collection, such as the need for biopsy from diseased tissue, collection at specific stages of menstruation, or rely on changes in regulatory patterns (e.g., gene expression, DNA methylation) induced by inflammation. Inflammation-induced changes in regulatory patterns may overlap with other gynecological disorders [10, 17], which may increase the likelihood of false-positive detection. Thus, an ideal biomarker would be detectable in healthy cells or body fluids and independent of transient disease, inflammation-induced, or cyclic physiological changes.

[0007] The mitochondrial genome represents a less explored biomarker repository. As shown in Figure 1, the mitochondrial genome encodes a complement of 24 genes, including 2 rRNAs and 22 tRNAs that ensure accurate translation of the remaining 13 genes essential for electron transport. Targeting mitochondrial DNA (mtDNA) is attractive from a diagnostic perspective due to its high mutation frequency, limited DNA repair capacity, presence in all nucleated cells, and high copy number (thousands of genomes per cell)

[27] . As a result, even low-frequency mutations and deletion events can be reliably amplified from the heteroplasmic mitochondrial population. Indeed, mtDNA mutations have been well-described as biomarkers for several cancers across multiple body sites, including bone, brain, breast, lung, colorectal, stomach, ovarian, prostate, and endometrial tissues [28–37]. Whereas the nuclear genome contains more than 3 billion base pairs, the mitochondrial (mt) genome is relatively small, containing 16,569 nucleic acid base pairs. Moreover, given the clonal expansion of mitochondria within the egg once fertilization has occurred, typically all mtDNA genomes in a given individual are identical. The mt genome is also unusual in that it is a circular, intronless DNA molecule interspersed with repeat motifs flanking sequences of specific length. Sequences between these repeats are prone to deletion under circumstances that are not well understood. Moreover, such deletions often include at least a portion of one or both of the flanking repeat sequences. As discussed further below, once the sequences constituting a deletion are removed, the remaining "parent" mtDNA recircularizes to form a "major sublimon." Similarly, deleted sequences may also recircularize to form a "minor sublimon." Given the number of repeats in the mt genome, there are many possible deletions. One of the best-known examples of these deletions is the 4977 bp "common deletion," which has been associated with a variety of pathologies. The common deletions have also been investigated as markers for endometriosis

[54] , but the lack of specificity did not suggest that such deletions are valid markers for the disease.Certain mitochondrial DNA deletions have previously been associated with several specific conditions and age-related disorders (see

[59] –

[64] ). A deletion of 8686 bp between nucleotides 5371 and 14058 of the mtDNA genome has also been published (

[65] ), but no correlation with disease stage or condition has been found.

[0008] In some cases, mtDNA deletions and other large-scale mtDNA rearrangements can result in transcribable mutant mtDNA sequences, resulting in mitochondrial fusion transcripts. Examples of the association between mitochondrial fusion transcripts and disease stages are described, for example, in the applicant's previous application numbers: PCT / CA2006 / 000652; PCT / CA2007 / 001711; PCT / CA2009 / 000351; and PCT / CA2010 / 000423, the entire disclosures of which are incorporated herein by reference.

[0009] During studies of endometrial cancer, mtDNA alterations in the endometrium have been detected [37-40]. However, these studies did not reveal consensus regions within the mtDNA genome or specific mtDNA alterations that correlate with endometrial disease. As a result, these studies did not suggest the conclusion that mtDNA alterations can be used as biomarkers for the detection of endometriosis. Furthermore, previous studies do not appear to have led to an association between mitochondrial fusion transcripts and endometrial disease or stages.

[0010] Thus, there is a need for accurate and / or more effective means of detecting endometrial diseases and / or conditions that address at least one deficiency in known methods.

[0011] [Summary of explanation] In one embodiment, the present description provides methods, reagents, and / or kits for detecting, diagnosing, and / or monitoring endometriosis in a subject. The present description includes the use of mitochondrial DNA (mtDNA) biomarkers, their fusion transcripts and / or translated fusion proteins, identified herein as associated with endometriosis. The method of the present invention can be performed using biological samples obtained from the subject to be screened. Such samples can include tissue (e.g., biopsy tissue), menstrual fluid, circulating blood, or blood derivatives such as serum or plasma. The currently described method can be performed on samples obtained non-invasively from a subject and serves as an effective means of determining whether a subject is suspected of having or exhibiting endometriosis and whether further invasive diagnostic investigation is mandatory.

[0012] In one embodiment, a method for detecting, diagnosing, and / or monitoring endometriosis in a mammalian subject is provided, the method comprising identifying, in a biological sample from the subject, an abnormal mitochondrial DNA (mtDNA) molecule having at least one deletion that results in a junction point in a rejoined or recircularized mtDNA nucleotide sequence, the junction point being between nucleotide pairs 8469:13447 of the mtDNA nucleotide sequence of SEQ ID NO:1, at nucleotide pair 7992:15730, nucleotide pair 9191:12909, nucleotide pair 9188:12906, nucleotide pair 10367:12829, nucleotide pair 6260:12814, nucleotide pair 7973:9023, nucleotide pair 9086:10313, nucleotide pair 9079:14988, nucleotide pair 7260:15540, nucleotide pair 8431:10841, nucleotide pair 8984:13833 or nucleotide pair 5362:14049.

[0013] In one embodiment, the method involves identifying the aberrant mtDNA by contacting the biological sample with a DNA probe or primer designed to hybridize to the aberrant mtDNA.

[0014] In one embodiment, the method includes identifying a fusion transcript of an aberrant mtDNA molecule.

[0015] In another embodiment, the method includes identifying a fusion protein encoded by an aberrant mtDNA molecule.

[0016] In one aspect, a method is provided for identifying an aberrant mitochondrial DNA (mtDNA) molecule having a deletion in a biological sample from a mammalian subject, the deletion comprising a nucleotide sequence between nucleotides 5362-14049; nucleotides 8469-13447; nucleotides 7992-15730; nucleotides 9191-12909; nucleotides 9188-12906; nucleotides 10367-12829; nucleotides 6260-12814; nucleotides 7973-9023; nucleotides 9086-10313; nucleotides 9079-14988; nucleotides 7260-15540; nucleotides 8431-10841; or nucleotides 8984-13833 of the mtDNA nucleotide sequence of SEQ ID NO:1, wherein once recircularized, the mtDNA comprises a junction point.

[0017] In another embodiment, a method is provided for identifying an abnormal mitochondrial DNA (mtDNA) molecule having a deletion in a biological sample from a mammalian subject, wherein once recircularized, the mtDNA comprises a junction point consisting of a first and a second nucleotide, with respect to SEQ ID NO:1: a) the deletion includes nucleotides 5377 to 14048, the first nucleotide being between nucleotides 5362 to 5377 and the second nucleotide being between nucleotides 14048 to 14063; b) the deletion includes nucleotides 8483 to 13446, the first nucleotide being between nucleotides 8469 to 8483 and the second nucleotide being between nucleotides 13446 to 13460; c) the deletion includes nucleotides 7993 to 15722, the first nucleotide being between nucleotides 7985 to 7993 and the second nucleotide being between nucleotides 15722 to 15730; d) the deletion includes nucleotides 9196 to 12908, the first nucleotide being between nucleotides 9191 to 9196 and the second nucleotide being between nucleotides 12908 to 12912; e) the deletion includes nucleotides 9196 to 12905, the first nucleotide being between nucleotides 9188 to 9196 and the second nucleotide being between nucleotides 12905 to 12913; f) the deletion includes nucleotides 10368-12825, wherein the first nucleotide is between nucleotides 10364-10368 and the second nucleotide is between nucleotides 12825-12829; g) the deletion comprises nucleotides 6261 to 12813, the first nucleotide being between nucleotides 6260 to 6271 and the second nucleotide being between nucleotides 12813 to 12824; h) the deletion includes nucleotides 7984 to 9022, the first nucleotide is between nucleotides 7973 to 7984 and the second nucleotide is between nucleotides 9022 to 9033; i) the deletion includes nucleotides 9087 to 10303, the first nucleotide being between nucleotides 9077 and 9087 and the second nucleotide being between nucleotides 10303 and 10313; j) the deletion includes nucleotides 9086 to 14987, wherein the first nucleotide is between nucleotides 9079 and 9086 and the second nucleotide is between nucleotides 14987 and 14904; k) the deletion includes nucleotides 7261 to 15531, wherein the first nucleotide is between nucleotides 7252 and 7261 and the second nucleotide is between nucleotides 15531 and 15540; l) the deletion comprises nucleotides 8440 to 10840, wherein the first nucleotide is between nucleotides 8431 to 8440 and the second nucleotide is between nucleotides 10840 to 10849; or m) the deletion comprises nucleotides 8994 to 13832, wherein the first nucleotide is between nucleotides 8984 and 8994 and the second nucleotide is between nucleotides 13832 and 13842.

[0018] In another embodiment, methods are provided for detecting fusion transcripts and fusion proteins resulting from aberrant mtDNA molecules or mtDNA deletions.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS Features of particular embodiments will become more apparent in the following detailed description, which refers to the accompanying drawings.

[0020] FIG. 1 is a diagram showing mitochondrial coding genes.

[0021] Figures 2A-2J show detection of fusion transcripts 1, 4, 14, 16, 120, 122, 193, 400, 516, and 586 in endometrial tissues as discussed in Example 1. The scatter plots represent normalized results of endometrial control tissues and endometriosis-positive tissues tested against probes specific for the 10 fusion transcripts (distinguished as transcript numbers 1 (Figure 2A); 4 (Figure 2B); 14 (Figure 2C); 16 (Figure 2D); 120 (Figure 2E); 122 (Figure 2F); 193 (Figure 2G); 400 (Figure 2H); 516 (Figure 2I); and 586 (Figure 2J)). The y-axis of each figure shows normalized relative luminescence units RLU (log2LOQProbe-Log2LOQHK23), where HK23 is the nuclear housekeeper transcript human β-2-microglobulin. The x-axis of each figure indicates histological diagnosis as determined by physician diagnosis at the time of laparoscopy, with endometriotic control=0.0 and endometriosis positive=1.0.

[0022] 3 shows the mtDNA genome, gene location, and fusion transcript map of mtDNA showing the locations of the ten portions of mtDNA that were deleted (i.e., "probes" or "targets") as described herein in SEQ ID NO: 1. The locations are indicated by lines spanning the length of each deletion.

[0023] Figures 4A and B show the diagnostic accuracy of the 1.2 kb and 3.7 kb deletions of Example 2, comparing symptomatic control samples and samples from patients with confirmed endometriotic disease. The 1.2 kb and 3.7 kb deletions were evaluated for their ability to distinguish symptomatic patient samples from samples from patients with confirmed endometriosis (all subtypes / stages combined). Receiver operator characteristic curves were generated and the area under the curve was calculated. Abbreviations: CI = confidence interval; ROC = receiver operator characteristic; Std = standard; vs = paired.

[0024] Figures 5A-5D show the diagnostic accuracy of the 1.2 kb deletion of Example 2 in discriminating between symptomatic control samples and samples of different endometriotic disease subtypes. The 1.2 kb deletion was evaluated for its ability to discriminate between symptomatic patient samples and samples from patients stratified by endometriosis subtype (peritoneal, ovarian, deep infiltrating). Figure 5A shows the distribution of the normalized 1.2 kb deletion for samples from symptomatic controls and patients with peritoneal, ovarian or deep infiltrating endometriosis. The box boundaries are within 25 th and 75 th percentiles, the central line represents the median, and the whiskers represent the 90th (top) and 10th (bottom) percentiles. Dots represent outliers (left). Descriptive statistics are summarized for each group (right). In Figures 5B-5D, receiver operator characteristic curves for the 1.2 kb deletion were constructed and the area under the curve indicating diagnostic accuracy was calculated. Abbreviations: CI = confidence interval; Dev = deviation; DIE = deep infiltrating endometriosis; N = number of specimens in each group; ROC = receiver operator characteristic; Std = standard; vs = paired.

[0025] Figures 6A-6D show the diagnostic accuracy of the 3.7 kb deletion of Example 2 in distinguishing between symptomatic control samples and samples of endometriotic disease subtypes. The 3.7 kb deletion was evaluated for its ability to distinguish between symptomatic patient samples and samples from patients stratified by endometriosis subtype (peritoneal, ovarian, deep invasive). Figure 6A shows the distribution of the normalized 3.7 kb deletion for sample samples from symptomatic controls and patients with peritoneal, ovarian or deep invasive endometriosis. Box boundaries represent the 25th and 75th percentiles, the center line represents the median, and whiskers represent the 90th (top) and 10th (bottom) percentiles. Dots represent outliers (left). Descriptive statistics are summarized for each group (right). In Figures 6B-6D, the receiver operator characteristic curve for the 3.7 kb deletion was constructed, and the area under the curve was calculated to show the diagnostic accuracy. Abbreviations: CI = confidence interval; Dev = deviation; DIE = deep infiltrating endometriosis; N = number of samples in each group; ROC = receiver operator characteristic; Std = standard; vs = paired.

[0026] Figures 7A-7C show the diagnostic accuracy of the 1.2 kb deletion of Example 2 in distinguishing between symptomatic control samples and samples from patients with known disease stages. The 1.2 kb deletion was evaluated for its ability to distinguish between symptomatic patient samples and samples from patients stratified by endometriosis stage (low or high). Figure 7A shows the distribution of the normalized 1.2 kb deletion for symptomatic controls and samples from patients with low (I / II) or high (III / IV) stages of endometriosis. Box boundaries are within 25 th Percentiles and 75 th Percentiles are shown, the middle line represents the median, and the whiskers represent the 90th percentile (top) and 10th percentile (bottom). Dots represent outliers (left). Descriptive statistics are summarized for each group (right). In Figures 7B and 7C, receiver operator characteristic curves were generated for the 1.2 kb deletion and the area under the curve, which indicates diagnostic accuracy, was calculated. Abbreviations: CI = confidence interval; Dev = deviation; N = number of samples in each group; ROC = receiver operator characteristic; Std = standard; vs = paired.

[0027] Figures 8A-8C show the diagnostic accuracy of the 3.7 kb deletion of Example 2 in distinguishing between symptomatic control samples and samples from patients with known disease stages. The 3.7 kb deletion was evaluated for its ability to distinguish between symptomatic patient samples and samples from patients stratified by endometriosis stage (low or high). Figure 8A shows the distribution of the normalized 3.7 kb deletion for samples from symptomatic controls and patients with low (I / II) or high (III / IV) stages of endometriosis. Box boundaries are within 25 th Percentiles and 75 th Percentiles represent the mean mean, the middle line represents the median, and whiskers represent the 90th (top) and 10th (bottom) percentiles. Dots represent outliers (left). Descriptive statistics are summarized for each group (right). In Figures 8B and 8C, receiver operator characteristic (ROC) curves were generated for the 3.7 kb deletion and the area under the curve, which indicates diagnostic accuracy, was calculated. Abbreviations: CI = confidence interval; Dev = deviation; N = number of samples in each group; ROC = receiver operator characteristic; Std = standard; vs = paired.

[0028] FIG. 9 is a scatter plot showing the difference in 8.7 kb deletion scores between endometriosis positive samples, symptomatic control samples and normal healthy control samples.

[0029] FIG. 10 is a box plot showing the difference in 8.7 kb deletion scores between endometriosis positive samples, symptomatic control samples and normal healthy control samples.

[0030] FIG. 11 shows the ROC curve for the 8.7 kb deletion comparing endometriosis positive patients versus healthy / normal controls.

[0031] Figure 12 shows the diagnostic accuracy of the 8.7 kb deletion - symptomatic vs. all endometriotic disease. The 8.7 kb deletion was assessed for its ability to distinguish between samples from symptomatic patients and those with confirmed endometriosis (all subtypes / stages combined) by calculating the area under the ROC curve. Abbreviations: CI = confidence interval; ROC = receiver operator characteristic; Std = standard; vs = versus.

[0032] Figures 13A-13B further show the diagnostic accuracy of the 8.7 kb deletion - control vs. disease - by subtype. These figures show a study of whether the 8.7 kb deletion assay can distinguish samples from symptomatic participants from those stratified by endometriosis subtype (peritoneal, ovarian, deep invasive). Figure 13A shows the distribution of the normalized 8.7 kb deletion for specimens from asymptomatic and symptomatic control, peritoneal, ovarian or deep invasive endometriosis participants. Box boundaries represent the 25th and 75th percentiles, the middle line represents the median, and whiskers represent the 90th (top) and 10th (bottom) percentiles. Dots represent outliers (left). Descriptive statistics are also summarized for each group. Figures 13B-13D show the area under the ROC curve, calculated to indicate diagnostic accuracy. Abbreviations: As Con=asymptomatic controls; CI=confidence interval; Dev=deviation; DIE=deep infiltrating endometriosis; N=number of specimens in each group; ROC=receiver operator characteristic; Sym Con=symptomatic controls; Std=standard; vs=pairs.

[0033] Figures 14A-14C further show the diagnostic accuracy of the 8.7 kb deletion - controls vs. disease by stage. These figures show whether the 8.7 kb deletion assay can distinguish between samples from symptomatic participants and those stratified by endometriosis stages I / II and III / IV. Figure 14A shows the distribution of the normalized 8.7 kb deletion for specimens from symptomatic controls, participants with low (I / II) or high (III / IV) endometriosis stages. Box boundaries represent the 25th and 75th percentiles, the middle line represents the median, and whiskers represent the 90th (top) and 10th (bottom) percentiles. Dots represent outliers (left). Descriptive statistics are summarized for each group. Figures 14B and 14C show the area under the ROC curve, calculated to indicate diagnostic accuracy. Abbreviations: CI = confidence interval; Dev = deviation; N = number of samples in each group; ROC = receiver operator characteristic; Sym Con = symptomatic control; Std = standard; vs = paired.

[0034] Figure 15 further illustrates the disease specificity of the 8.7 kb deletion to endometriosis. This figure summarizes the evaluation of the frequency of the 8.7 kb deletion in female cancers, including endometrial, ovarian and breast cancer. Standardized distribution of the 8.7 kb deletion for samples from endometrial, ovarian, breast cancer, symptomatic controls, and participants with peritoneal, ovarian or deep infiltrating endometriosis. Box boundaries represent the 25th and 75th percentiles, center line represents the median, and whiskers represent the 90th (top) and 10th (bottom) percentiles. Dots represent outliers (left).

[0035] FIG. 16 is a scatter plot showing the difference in scores of the 4.8 kb deletion between endometriosis positive samples, symptomatic control samples and normal healthy control samples.

[0036] FIG. 17 is a box plot showing the difference in 4.8 kb deletion scores between endometriosis positive samples, symptomatic control samples and normal healthy control samples.

[0037] FIG. 18 shows the ROC of the 4.8 kb deletion comparing data from endometriosis positive patients and symptomatic controls.

[0038] FIG. 19 shows the ROC of the 4.8 kb deletion comparing data from endometriosis positive patients versus healthy / normal controls.

[0039] FIG. 20 shows the occurrence of deletions according to the present invention.

[0040] Detailed Description Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Suitable materials and methods for the practice or testing of the present invention are described below, although other known materials and methods similar or equivalent to those described herein can be used.

[0041] As used herein with respect to mtDNA, the terms "deletion," "deletion fragment," or "deletion sequence" are understood to mean a nucleotide sequence or segment that is removed or deleted from a wild-type or naturally occurring mtDNA genome.

[0042] The term "wild-type mtDNA" or "naturally occurring mtDNA" refers to the revised Camcridge Reference Sequence (rCRS) (2001, GenBank Accession Number: NC_012920.1), which is provided herein as SEQ ID NO: 1. This sequence is identified as being 16569 bp in length, although the actual number of nucleotides is 16568. As is known in the art, this sequence contains a gap or placeholder nucleotide at position 3107.

[0043] As used herein with respect to mtDNA, the terms "mutation" or "abnormality" are understood to be synonymous with the term "deletion."

[0044] As used in the context of the present description, the terms "mutated mtDNA" or "abnormal mtDNA" are understood to mean an mtDNA molecule that has at least one deletion (as defined above) in its genomic sequence.

[0045] The term "junction" or "junction point" is understood to mean a position in the nucleotide sequence of a recircularized mtDNA molecule, which includes the rejoining or splicing of nucleotides of the remaining mtDNA genomic sequence following the removal of the deletion. As will be further discussed herein, the deletion event typically results in the creation of two new sequence fragments, consisting of the parent sequence corresponding to the mtDNA molecule rejoined after the removal of the deletion and the deleted sequence corresponding to the deleted portion. Generally, the parent sequence is longer than the deleted sequence. Often, and as discussed above, both the long and short fragments recircularize to form what are known as the major and minor sublimons, respectively. As will be understood, both sublimons have unique junction points in their nucleotide sequences. Thus, the term junction or junction point can be used to refer to either the major or minor sublimon.

[0046] The phrase "having a deletion" is understood to refer to an mtDNA molecule having a nucleotide sequence from which the deleted sequence is removed. In other words, the phrase "mtDNA having a deletion" refers to the parent nucleic acid. Thus, "mtDNA having a common deletion" refers to an mtDNA molecule having a sequence that does not include the 4977 bp deleted sequence.

[0047] As used herein, the term "detect" is understood to mean to determine or identify and / or measure or quantify the presence in a biological sample of a particular feature. In one embodiment, the term "detect" is used herein to refer to the identification of mitochondrial DNA (mtDNA) sequences, more particularly mtDNA having a deletion. The term "detect" may also be used to refer to the identification of mitochondrial fusion transcripts and / or proteins encoded by such mtDNA molecules. In the latter case, the protein is referred to herein as a "fusion protein" and contains an amino acid sequence resulting from translation of the rejoined mtDNA after the deletion event. Such mtDNA may contain the parent mtDNA, or an aberrant mtDNA or deleted sequence.

[0048] As used herein, the term "diagnose" is understood to mean the identification of disease state or disease stage, or the determination of high or increased probability of disease state or disease stage.For example, in the context of this description, when the mtDNA molecule or fusion transcript described herein is detected, the presence of endometriosis stage or condition is deemed to be higher or "diagnosed".It is understood that the actual diagnosis or clinical diagnosis of this stage or condition is made by clinicians upon examination of biopsy sample or other such means.Therefore, in some cases, the terms "detect" and "diagnose" can be used interchangeably herein.

[0049] As used herein, the term "biological sample" is understood to refer to tissue or bodily fluid containing cells or nucleic acids from which molecules of interest can be obtained. Biological samples can either be used directly as obtained from the source or can first be subjected to pretreatment to modify the characteristics of the sample. In one embodiment, the biological sample is blood, particularly circulating blood, and it is understood that the term "blood" as used herein is intended to include blood derivatives such as plasma and / or serum. In another embodiment, the biological sample is menstrual fluid, including menstrual blood. In another embodiment, the biological sample is a tissue sample obtained from a subject. In one embodiment, circulating blood can be used as a biological sample. It is understood that blood samples for the purposes of this description can be taken from any source on the subject's body. This includes, but is not limited to, blood taken from a venous source such as by a syringe, collection of a menstrual fluid sample, or capillary blood, such as blood taken by finger prick. Using the currently described method using circulating blood (including blood derivatives, as described above) provides an effective means of detecting the presence of endometriosis in individuals suspected of having such a condition without undergoing unnecessarily painful and dangerous invasive procedures. As described above, in situations where the currently described method suggests the presence of endometriosis, diagnosis still requires clinical evaluation and possibly laparoscopy / surgery or analysis of a biopsy sample. Thus, in one embodiment, it is understood that the currently described method, especially when using circulating blood (or one or more derivatives thereof, as described above) as a biological sample, can be performed on a subpopulation of patients, including those individuals who have one or more symptoms suggesting the presence of endometriosis. It is also understood that the currently described method may be performed on members of the general population as an initial step in screening for endometriosis. In other words, the currently described method may be performed on asymptomatic subjects (i.e., individuals who do not exhibit symptoms).

[0050] As used herein, the phrase "mitochondrial fusion transcript" or "fusion transcript" refers to an RNA transcript produced as a result of transcription of an mtDNA sequence.

[0051] As used herein, the term "mutant" refers to a nucleic acid sequence that differs from a naturally occurring sequence but retains its essential or functional properties. In one embodiment, the term "mutant" refers to a sequence that changes relative to a wild-type sequence. In general, in the case of mtDNA, a mutant is closely similar overall and in many regions identical to a selected mtDNA sequence. In the context of this description, a mutant may include at least one of the nucleotides of the junction point of a spliced ​​gene, and may further include one or more nucleotides adjacent thereto. In one embodiment, a mutant sequence is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a given mtDNA sequence described herein or its complementary strand.

[0052] As used herein, the phrase "substantially similar" refers to nucleic acids that are functionally the same but differ in their respective nucleic acid sequences.In one embodiment, two sequences that are substantially similar to each other may be called "variants".Thus, two nucleic acid molecules may be considered to be substantially similar if the difference of one or more nucleotides between the respective nucleic acid sequences does not change their functional properties or the functional properties of any polypeptide encoded by such nucleic acid.As will be understood, due to the degeneracy of the genetic code, base pair changes do not cause any changes in the encoded amino acid sequence.

[0053] The phrase "substantial complementarity" refers to a sufficiently high degree of complementarity between nucleotide sequences of nucleic acid molecules to allow hybridization between them, but not necessarily 100% complementarity. For example, a primer or probe having substantial complementarity to a target sequence may have 80% to 99% sequence identity to the target sequence. In one embodiment, as used herein, substantial complementarity refers to at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity between sequences.

[0054] As used herein, the term "fragment" refers to a nucleic acid sequence that is a portion of a given mitochondrial genome sequence or a complementary strand thereto. In one embodiment, such a "portion" includes at least two of the nucleotides that comprise the junction point of the spliced ​​gene, and may further include one or more nucleotides adjacent thereto. That is, the portion includes the recombined or recircularized DNA sequence after removal of the deletion. The fragments described herein are at least about 150 nucleotides (nt), at least about 75 nt, at least about 50 nt, at least about 40 nt, at least about 30 nt, at least about 20 nt, or preferably at least about 15 nt in length. Although certain minimum nucleotide lengths are listed above, it is understood that as described herein, fragments of any size (e.g., 50, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 6000, 7000, 8000 or more nucleotides) are also contemplated.

[0055] As used herein in the context of sequence length, the term "about" includes the specifically recited value or a few (5, 4, 3, 2, or 1) nucleotides greater or less at either or both termini.

[0056] As used herein, the term "probe" or "primer" refers to an oligonucleotide molecule that forms a duplex structure or "hybridizes" with a target nucleic acid due to the complementarity of a portion of the nucleotide sequence of the target molecule with at least a portion of the nucleotide sequence of the probe / primer. The target nucleic acid molecule may, in some cases, be a fragment of a naturally occurring nucleic acid molecule. The probes described herein may be labeled according to methods known in the art. It is understood that the probes or primers described herein are used under appropriate hybridization conditions as known to those skilled in the art. The probes herein may also be referred to as hybridizing probes. The probes and primers described herein may be of any length, as will be understood by those skilled in the art. By way of example only, the currently described probes and primers may have a length of about 150, 140, 130, 120, 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, or 10 nucleotides (nt). In a preferred embodiment, the probes and / or primers described herein are about 12 to about 35 nt in length, or preferably about 18 to about 25 nt in length, and more preferably about 15 nt in length. As will be appreciated by those skilled in the art, the probes may have a nucleotide length longer than the primers. Thus, in some cases, the probes described herein may have a length of about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, or 2500 nucleotides. This description is not limited to the length of any particular probe or primer.

[0057] The terms "comprise", "comprises", "comprised" or "comprising" may be used in this description. When used in this specification (including the specification and / or claims), these terms should be interpreted as specifying the presence of the stated features, integers, steps, or components. However, they do not exclude the presence of one or more other features, integers, steps, components, or groups thereof, as would be apparent to a person skilled in the relevant art. Thus, when used in this specification, the term "comprising" means "consisting at least in part of". When interpreting descriptions in this specification that include the term, all features prefaced by the term in the respective description must be present, but other features may also be present. Related terms such as "comprise" and "comprised" should be interpreted in the same manner.

[0058] The term "and / or" can mean "and" or "or."

[0059] Unless otherwise stated in this specification, the article "a" used to identify any element is not intended to constitute a limitation to only one, but instead is understood to mean "at least one" or "one or more."

[0060] As described herein, the inventors have identified novel mtDNA deletions, which in one embodiment are associated with endometriosis and therefore constitute accurate diagnostic markers for such conditions. The inventors have also identified novel mtDNA fusion transcripts, which in one embodiment are associated with endometriosis. Both of these embodiments are discussed further below. Translation products resulting from fusion transcripts are also encompassed by the present description.

[0061] In one aspect, the present invention relates to the inventors' hypothesis that endometrial cells that are shed into menstrual fluid during menstruation will have the same genetic profile as endometrioid cells present in ectopic and / or normal endometrial lesions.Using the knowledge gained from mapping large deletions in the human mitochondrial genome, the observation of high frequency of these deletions, and the evidence of transcriptionally activated mutant mtDNA molecules in other disease types, the inventors further hypothesized that mitochondrial deletions and fusion transcripts may be present in endometrioid cells present in ectopic and / or normal endometrioid lesions.

[0062] To test these hypotheses, 268 mitochondrial fusion transcripts were selected based on predicted direct and indirect repeats across the mitochondrial genome and screened for use as biomarkers for endometriosis. A number of mtDNA deletions and corresponding fusion transcripts were identified by the inventors as being particularly useful in distinguishing samples with endometriosis from those without endometriosis. These deletions and fusion transcripts are discussed further below. These mtDNA molecules produce fusion sequences that have an open reading frame (ORF) that can be transcribed by the mitochondrial transcription machinery, resulting in fusion transcripts. Protein products or fusion proteins encoded by such fusion transcripts are also expected to be produced.

[0063] <(1.0) mtDNA deletions, fusion transcripts, translation products> (1.1) Mitochondrial DNA (mtDNA) mutations As mentioned above, mtDNA mutations generally involve the deletion of a portion of the mtDNA wild-type sequence. The present explanation is based on the association of certain mtDNA mutations, specifically deletions of the mtDNA genomic sequence, with endometriosis.

[0064] According to this description, to determine the candidate genome sequence, the junction points resulting from the sequence deletion were first identified. The sequence deletion was mainly identified by direct or indirect repeat elements flanking the sequence to be deleted at the 5' and 3' ends. The removal of a portion of nucleotides from the genome and then ligation of the remaining genome creates a new junction point.

[0065] In identifying the junction point, the nucleotides of the gene adjacent to the junction point are determined to identify the spliced ​​gene.Typically, the spliced ​​gene contains the start codon from the first gene and the stop codon from the second gene, and can be expressed as a continuous transcript, i.e., it keeps the reading frame from the beginning to the end of both spliced ​​genes.It is also possible that another start codon or stop codon contained in the gene sequence can be used.

[0066] Large deletions in the mitochondrial genome often result in two products resulting from the mutation process. These products are the result of recircularization of both parts of the mtDNA genome: 1) a short sequence (which may correspond, in one embodiment, to the deleted mtDNA sequence) and 2) a long sequence (which may correspond, in one embodiment, to the remaining mtDNA genome sequence). It will be understood that, depending on the size of the deletion, the deletion may be larger than the remaining mtDNA. This situation occurs, for example, when the length of the deleted sequence is greater than about 8200 bp. Often, both the short sequence and the long sequence recircularize to form what are known as the minor and major sublimones, respectively. If the minor component is an insufficient number of nucleotides, recircularization is not possible, in which case the mutation process only results in the major sublimon. As discussed herein, both the major and minor sublimones can be identified, thereby allowing both molecules to be used to detect, diagnose, and / or monitor endometriosis.

[0067] ((1.2) Fusion transcripts) Large-scale rearrangement mutation in mitochondrial genome leads to the generation of fusion transcripts.Therefore, it is expected that mtDNA rearrangement related to endometriosis will lead to fusion transcripts related to endometriosis.Therefore, the use of mtDNA encoding such transcripts and the probe directed thereto for the diagnosis and monitoring of endometriosis is provided herein.

[0068] This description provides the identification of fusion transcripts and associated hybridization probes and primers that are useful in methods for predicting, diagnosing, and / or monitoring endometriosis.Those skilled in the art will understand that such molecules can be derived through the isolation of naturally occurring transcripts, or alternatively, by recombinant expression of mtDNA molecules isolated according to the methods of the present invention.As discussed, such mtDNA molecules typically contain spliced ​​genes that have the start codon from a first gene and the stop codon of a second gene.Therefore, the fusion transcripts derived therefrom contain the junction points associated with the spliced ​​genes.

[0069] (1.3) Translation products Based on the fusion transcripts described herein, the description also provides the amino acid sequence of the predicted protein resulting from translation of the subject fusion transcript, i.e., the "fusion protein." The description also provides the translation product of at least a portion of the fusion transcript, specifically the portion that includes the transcribed fusion site or junction point of mtDNA.

[0070] The fusion proteins of the present description can be recovered and purified from biological samples by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, hydrophobic charge interaction chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is used for purification.

[0071] Assaying fusion protein levels in biological samples can occur using a variety of techniques. For example, protein expression in tissues can be studied using classical immunohistological methods (Jalkanen et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol. 105:3087-3096 (1987)). Other methods useful for detecting protein expression include immunoassays such as enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs). Suitable antibody assay labels are known in the art and include enzyme labels (e.g., glucose oxidase), and radioisotopes (e.g., iodine ( <125> I, <121> I), Carbon ( <14> C), sulfur ( <35> S), tritium ( <3> H), Indium ( <112> In), and technetium (<99m>Tc), and fluorescent labels (e.g., fluorescein and rhodamine), and biotin.

[0072] The polypeptides of the present description may also be produced by recombinant techniques known in the art, which typically involve transformation (including transfection, transduction or infection) of a suitable host cell with an expression vector containing a polynucleotide encoding the protein or polypeptide of interest.

[0073] (Antibodies and Protein Binders) Protein-specific antibodies for use in the assays described herein can be raised against the wild-type or expressed fusion proteins described herein, or antigenic polypeptide fragments thereof, which can be presented in an animal system (e.g., rabbit or mouse) with a carrier protein (e.g., albumin), or without a carrier if it is long enough (at least about 25 amino acids). Although antibodies are described, it is understood that any other suitable binding agent specific for identifying the protein can also be used. In any case, the antibody or binding agent can identify the fusion proteins described herein by methods that specifically bind to regions of such proteins that represent or indicate the deletion. In one embodiment, the fusion protein has a unique amino acid profile that represents the translation of the junction point of the mtDNA molecule (either the major or minor sublimon) after the deletion event.

[0074] As used herein, the term "antibody" (Ab) or "monoclonal antibody" (Mab) is meant to include intact molecules and antibody fragments, or antigen-binding fragments thereof, such as Fab and F(ab')2 fragments, that are capable of specifically binding to, or have "specificity" for, mitochondrial fusion proteins. Fab and F(ab')2 fragments lack the Fc fragment of intact antibodies, clear more rapidly from the circulation, and may be less susceptible to non-specific tissue binding of intact antibodies (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). Thus, these fragments are preferred.

[0075] The antibodies of the present invention can be prepared by any of a variety of methods. For example, cells expressing the mitochondrial fusion protein or an antigenic fragment thereof can be administered to an animal to induce the production of serum containing polyclonal antibodies. In one method, a preparation of the mitochondrial fusion protein is prepared and purified to be substantially free of natural contaminants. Such a preparation is then introduced into an animal to produce polyclonal antisera of greater specific activity.

[0076] In a related method, the antibody of the present description is a monoclonal antibody. Such monoclonal antibody can be prepared using hybridoma technology (Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, NY, (1981) pp. 563-681). In general, such a procedure includes immunizing animals (preferably mice) with mitochondrial fusion protein antigen or mitochondrial fusion protein expressing cells.

[0077] In one embodiment, the description encompasses immunological assays using antibodies or antigen-binding fragments having specificity for the fusion proteins described herein (as described above). Such immunological assays may be facilitated by kits containing the antibodies or antigen-binding fragments along with any other necessary reagents, test strips, materials, instructions, etc.

[0078] Assay Measuring the level of translation products such as fusion proteins in biological samples can determine the presence or progression of endometriosis in a subject.Thus, in one embodiment, the present description provides a method for predicting, diagnosing or monitoring endometriosis, which includes obtaining one or more biological samples, extracting mitochondrial fusion proteins from the samples, and assaying the samples for one or more molecules by quantifying the amount of such molecules in the samples and comparing the detected amount with a reference value.As will be understood by those skilled in the art, the reference value is based on whether the method is intended to predict, diagnose or monitor endometriosis.Thus, the reference value can be related to protein data collected from one or more control samples or biological samples that are not positive for endometriosis, protein data collected from one or more biological samples that are positive for endometriosis, and / or protein data collected from one or more biological samples taken over time.

[0079] Techniques for quantifying protein in a sample are well known in the art and include, for example, classical immunohistological methods (Jalkanen et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol. 105:3087-3096 (1987)). Additional methods useful for detecting protein expression include immunoassays, such as radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA).

[0080] In one aspect, the description provides a method of detecting, diagnosing or monitoring endometriosis in a mammal, comprising assaying a tissue sample from the mammal for the presence of at least one mitochondrial fusion protein.

[0081] <(2.0) Probes and primers> (2.1) mtDNA Probes and Primers Also described herein are mtDNA hybridization probes and / or primers that can hybridize to the aberrant mtDNA sequences under suitable hybridization conditions. Any known hybridization method can be used.

[0082] Probes and / or primers can be generated directly against the exemplary mtDNA fusion molecules described herein (e.g., those listed in Table 1 below), or against fragments or variants thereof. For example, the aberrant mtDNA sequences discussed herein can be used to design primers or probes that detect nucleic acid sequences that contain the fusion nucleotide sequence of interest. As will be understood by those of skill in the art, primers and / or probes that hybridize to these nucleic acid molecules can hybridize under highly stringent hybridization conditions or under low stringency conditions. Such conditions are known to those of skill in the art and are described, for example, in Current Protocols in Molecular Biology (John Wiley & Sons, New York (1989)), 6.3.1-6.3.6.

[0083] In some embodiments, the probes and primers described herein contain sequences complementary to at least a portion of the aberrant mtDNA that contains the junction point of the spliced ​​gene. As stated above, this "portion" includes at least two nucleotides remaining in the mtDNA genome after removal of the deletion, thereby resulting in the junction point. The junction point is identified herein as A:B, with "A" and "B" representing the mtDNA genomic nucleotides on opposite sides of the deleted sequence, but adjacent to each other after the remaining sequence is recircularized. The "portion" may further include one or more nucleotides adjacent to the junction point. In this regard, the description encompasses any suitable targeting mechanism that uses the nucleotides involved and / or adjacent to the junction point A:B to select mtDNA molecules. It is further contemplated herein that the sequences of the primers and probes may be modified by one or more base pairs while still allowing hybridization to the target sequence. Such primers or probes are said to have "substantial complementarity" to the target sequence. As mentioned above, after a deletion event, both a major and a minor sublimon may result, and both such sublimons will have their respective junction points as defined above once the molecule is recircularized.

[0084] Additionally, the description encompasses, in one embodiment, primers designed to span the deletion junction or junction point A:B in either a forward or reverse orientation. In another embodiment, one or more primers may be designed to hybridize to a position on the target sequence adjacent to the junction point.

[0085] Various types of probes known in the art are contemplated for use in this description. For example, the probe can be a hybridization probe, and its binding to the target nucleotide sequence can be detected using common DNA binding dyes such as ethidium bromide, SYBR® Green, SYBR® Gold, etc. Alternatively, the probe can incorporate one or more detectable labels. The detectable label is a molecule or a molecule that can be detected directly or indirectly, and is selected so that the ability of the probe to hybridize to its target sequence is not affected. Methods for labeling nucleic acid sequences are well known in the art (see, for example, Ausubel et al, (1997 & updated) Current Protocols in Molecular Biology, Wiley & Sons, New York).

[0086] Labels suitable for use with the probes of the present description include those that can be directly detected, such as radioisotopes, fluorophores, chemiluminophores, enzymes, colloidal particles, fluorescent microparticles, etc. Those skilled in the art will understand that directly detectable labels may require additional components, such as substrates, triggering reagents, light, etc., to enable detection of the label. The present description also contemplates the use of labels that are indirectly detected.

[0087] As stated above, the presently described probes and primers can be of any suitable length, as would be understood by one of skill in the art. The nucleotide lengths of the probes and primers of this description have been discussed above. As stated above, the probes and / or primers described herein can be preferably from about 12 to about 25 nucleotides in length, more preferably from about 12 to about 15 nt in length. It is understood that the primers and / or probes described herein can be preferably at least as long as the size of an mtDNA repeat (i.e., repeated) sequence. This description is not limited to the length of any particular primer or probe.

[0088] The probes described herein preferably hybridize to the nucleic acid molecules from the biological samples described herein, thereby enabling the methods described.Thus, in one embodiment, a hybridization probe for use in detecting endometriosis is provided, which is complementary or substantially complementary to at least a portion of the abnormal mtDNA molecule described herein or a portion of the deleted sequence from the mtDNA genome.

[0089] (2.2) Fusion Transcript Probes and Primers Once the fusion transcripts are characterized, primers or probes can be developed to target the transcripts in biological samples. Such primers and probes can be prepared using any known method (as described above) or as described in the examples provided below. Probes can be generated, for example, for the fusion transcripts, and detection techniques (e.g., QuantiGene™ 2.0 by Panomics™) can be used to detect the presence of the transcripts in the sample. Primers and probes can be generated directly for the exemplary fusion transcripts described herein, or for fragments or variants thereof. For example, the sequences described herein (e.g., sequences listed in Table 2 below) can be used to design probes or primers that detect RNA sequences containing the fusion sequence of interest.

[0090] As will be understood by those skilled in the art, the probes and primers designed to hybridize to the fusion transcripts described herein contain sequences that are complementary or substantially complementary to at least a portion of the transcript expressing the junction point of the spliced ​​gene. This portion includes at least two of the nucleotides complementary to the expressed junction point, and may further include one or more complementary nucleotides adjacent thereto. In this regard, the present description encompasses any suitable targeting mechanism that selects for fusion transcripts that use nucleotides involved and adjacent to the junction point of the spliced ​​gene.

[0091] Various types of probes and labeling methods known in the art are contemplated for the preparation of the transcript probes described herein. Some examples of such types and methods are described above with respect to the detection of genomic sequences. The transcript probes of this description are at least about 150 nt, at least about 75 nt, at least about 50 nt, at least about 40 nt, at least about 30 nt, at least about 20 nt, or preferably at least about 12-15 nt in length. A probe that is "at least 20 nt in length" is intended to include, for example, 20 or more consecutive bases that are complementary to the mtDNA sequence of the present invention. Of course, larger probes (e.g., 50, 150, 500, 600, 2000 nucleotides) are preferred. As described above, primers or probes of 18-25 nt are preferred.

[0092] In some embodiments, one or more hybridization probes and / or primers for use in detecting endometriosis are provided, wherein the one or more probes and / or primers are complementary or substantially complementary to at least a portion of a mitochondrial fusion transcript described herein.

[0093] <(3.0) Assays for detecting mtDNA deletions, fusion transcripts and their protein products> As mentioned above, the present description provides mitochondrial DNA biomarkers that are useful in detecting, diagnosing, and / or monitoring endometriosis in a subject using a biological sample derived from the subject. Specifically, such biological sample is non-invasively collected menstrual fluid, circulating blood, and / or tissue (such as biopsy tissue). The present description thus provides, in one embodiment, a test based on menstrual fluid or blood that allows for early and accurate detection of endometriosis, thereby preventing unnecessary initial and repeated surgical procedures. Thus, the method described herein reduces the need for unnecessary laparoscopic procedures when endometriosis is suspected but not detected. The present method also assists in determining whether endometriosis has recurred by allowing observation of endometriosis in a subject over time.

[0094] ((3.1) Measurement of abnormal mtDNA) According to the methods described herein, measuring the level of one or more abnormal mtDNA markers of the present invention in a biological sample can determine the presence or stage or progression of endometriosis in a subject. The present description thus provides a method for detecting, diagnosing and / or monitoring endometriosis in a subject, which comprises assaying a biological sample from the subject for one or more abnormal mtDNA biomarkers (i.e., "markers") as described herein by measuring and / or quantifying the amount of one or more abnormal mtDNA markers in the sample. Once quantified, the amount of the marker can be compared to a reference value (i.e., a control). The reference value may be based on whether the method is intended to detect, diagnose or monitor endometriosis. For example, when detecting or diagnosing endometriosis, the reference value may include the amount of abnormal mtDNA in a sample from a healthy subject, i.e., a subject not suffering from endometriosis. Such a sample may be described herein as a "known non-endometriotic" (i.e., "unrelated") biological sample. Alternatively, the reference value may include the amount of abnormal mtDNA in a sample from a subject known to be afflicted with endometriosis. Such a sample may be described herein as a "known endometriotic" (i.e., "related") biological sample. If the control includes a value or amount from a non-endometriotic source, it may be described herein as a "non-endometriotic amount." In other embodiments described herein, the control may include a reference value of another specimen from the same biological sample. In some cases, as further described herein, the amount of abnormal mtDNA may be first normalized to the amount of nuclear DNA (e.g., encoding one or more housekeeping genes, e.g., genes encoding rRNA) taken from the same subject. In one embodiment, the nuclear DNA sequence used may encode 18S rRNA. The normalized value of mtDNA may then be compared to a threshold value. When detecting or diagnosing endometriosis, an increased amount of abnormal mtDNA in a subject is indicative of endometriosis.When monitoring for endometriosis, biological samples can be taken from a subject over time and compared over a period of time. An increase over time in the amount of one or more of the abnormal mtDNAs described herein indicates the onset, recurrence or progression of endometriosis in the subject.

[0095] The presently described methods also include assaying a biological sample for a panel of aberrant mtDNA markers described herein, such a panel including two or more subject mtDNA markers. For example, such a panel may include two, three, four, five, six, seven, eight, nine, ten, or more of the presently described mtDNA markers.

[0096] In one embodiment, provided herein is a method of detecting endometriosis in a mammal, comprising assaying a biological sample (e.g., blood, menstrual fluid, tissue sample, etc.) from a mammalian subject for the presence of abnormal mtDNA by hybridizing the sample with at least one hybridization probe capable of recognizing or hybridizing to a mutant mtDNA sequence as described herein. Specifically, as described herein, such probes are provided with nucleotide sequences adapted to hybridize with a portion of the mtDNA molecule of the sample, such portion including a junction point as described herein.

[0097] In some embodiments, the method comprises assaying a biological sample from a mammal by hybridizing the sample with at least two primers adapted to hybridize to abnormal mtDNA molecules as described herein. In one embodiment, one of the primers can be designed with a nucleotide sequence that is complementary to a portion of the mtDNA having a junction point as described herein. In another embodiment, the primer can be provided with a nucleotide sequence that hybridizes to a region adjacent to the junction point of the mtDNA and adapted to overlap the junction point.

[0098] In another aspect, the present description provides a method for detecting endometriosis, the assay comprising: a) performing a hybridization reaction using at least one probe described herein to enable the at least one probe to hybridize to an abnormal mitochondrial DNA sequence extracted from a biological sample; b) quantifying the amount of at least one aberrant mitochondrial DNA sequence in the sample by quantifying the amount of mitochondrial DNA hybridized to at least one probe; and c) comparing the amount of mitochondrial DNA in the sample to at least one known reference value, - if the baseline contains an amount of mtDNA not associated with endometriosis, a higher amount of abnormal mtDNA in the sample indicates the presence of endometriosis; or - If the baseline includes amounts of mtDNA associated with endometriosis, then a lower amount of abnormal mtDNA in the sample indicates the absence of endometriosis.

[0099] Methods and screening means for diagnosing endometriosis by identifying specific mitochondrial mutations are also contemplated herein.Any known hybridization method can be used to carry out such methods, including but not limited to branched DNA and qPCR, probe and / or primer-based techniques, both singleplex and multiplex.Array technology can also be used, with oligonucleotide probes that match wild-type or mutated regions, and control probes.Commercially available arrays, such as microarrays or gene chips, are suitable for use in the methods described herein.

[0100] Thus, by detecting the abnormal mtDNA molecules described herein in a biological sample, it is possible to detect or diagnose endometriosis in a subject.Furthermore, by measuring and comparing the amount of abnormal mtDNA in successive samples from a subject over time, either qualitatively or quantitatively, the progression of endometriosis in such a subject can be monitored.

[0101] ((3.2) Measurement of Fusion Transcripts) Measuring the level of the mitochondrial fusion transcripts described herein in a biological sample can also determine the presence or stage or progression of endometriosis in a subject. Thus, a method for detecting, diagnosing and / or monitoring endometriosis is provided, which comprises extracting mitochondrial RNA from one or more biological samples obtained from a subject, and assaying the sample for fusion transcripts corresponding to the abnormal mtDNA described herein. Such an assay can comprise quantifying the amount of one or more fusion transcripts in the sample and comparing the detected amount to a reference value. The reference value is based on whether the method is intended to diagnose or monitor endometriosis. Thus, the reference value can relate to transcript data collected from one or more known non-endometriosis biological samples, from one or more known endometriosis biological samples, from a population of known non-endometriosis or known endometriosis samples, and / or from one or more biological samples taken from a subject over time.

[0102] In one embodiment, the methods described herein involve assaying one or more biological samples from a subject for a panel of fusion transcript markers indicative of endometriosis, the panel comprising 2, 3, 4, 5, 6, 7, 8, 9 or 10 RNA markers described herein.

[0103] Thus, in one aspect, a method for detecting endometriosis in a mammal is provided, the method comprising assaying a biological sample (e.g., blood, menstrual fluid, or tissue) from the mammal for the presence of at least one fusion transcript described herein by hybridizing the sample with at least one hybridization probe having a nucleic acid sequence complementary to at least a portion of the mitochondrial fusion transcript, which portion includes a fusion junction in the mitochondrial fusion transcript.

[0104] In another embodiment, a method is provided that comprises assaying a biological sample from a mammal by hybridizing the sample with at least two primers.As described above, at least one of the primers can have a sequence that allows hybridization to a portion of the fusion transcript that includes the fusion junction.In another embodiment, the primer can have a sequence that allows hybridization to a flanking region of the fusion junction.

[0105] In another embodiment, the present invention provides a method as described above, wherein the assay comprises: a) performing a hybridization reaction with at least one of the above-mentioned probes, allowing the at least one probe to hybridize to a complementary mitochondrial fusion transcript; b) quantifying the amount of at least one mitochondrial fusion transcript in the sample by quantifying the amount of transcript hybridized to at least one probe; and c) comparing the amount of mitochondrial fusion transcripts in the sample with at least one known reference value, wherein: - if the reference value includes an amount of a mitochondrial fusion transcript not associated with endometriosis, a higher amount thereof in the sample indicates the presence of endometriosis; and - If the baseline contains an amount of mtDNA associated with endometriosis, then a lower amount of abnormal mtDNA in the sample indicates the absence of endometriosis.

[0106] ((3.3) Detection of translated proteins) The translation products, proteins, of the fusion transcripts described herein can be detected using commonly known methods, such as immunological assays utilizing antibodies or other such specific binding moieties, which specifically bind to the translated fusion site or junction of mtDNA.

[0107] <(4.0) Kit> The present description encompasses diagnostic or screening kits for the in vitro detection, diagnosis, and / or monitoring of endometriosis in a subject. Such kits preferably include one or more probes or primers as described herein, optionally in combination with reagents, instructions, tools, and / or containers, etc., as may be required to perform the assay.

[0108] The kits may include reagents necessary to perform the diagnostic assays, such as buffers, salts, detection reagents, anticoagulants, etc. Other components, such as buffers and solutions for isolation and / or processing of biological samples, may also be included in the kits. One or more of the components of the kit may be lyophilized, and the kit may further include reagents suitable for reconstitution of the lyophilized components.

[0109] Optionally, the kits described herein may also include sampling means, reaction vessels, mixing vessels, and / or other components to facilitate collection and / or preparation of test samples. The kits may also optionally include instructions for use, which may be provided in paper form or in computer readable form such as a disk, CD, DVD, etc.

[0110] In one embodiment, the present description provides a kit for performing an in vitro assay for detecting and / or diagnosing endometriosis comprising a hybridization probe described herein and at least one reagent for performing the assay.

[0111] In one embodiment, the kit described herein comprises at least one hybridization probe that is complementary to at least a portion of the abnormal mtDNA described herein or at least a portion of the mitochondrial RNA fusion transcript described herein.As described above, in one embodiment, the portion of the sequence that the probe hybridizes comprises the junction point or fusion junction in the mtDNA or fusion transcript.In one embodiment, the kit may comprise one or more probes that are adapted to hybridize to one or more control sequences.

[0112] In another embodiment, the kit described herein comprises a pair of primers (e.g., forward and reverse primers) for amplifying at least a portion of the aberrant mtDNA described herein or at least a portion of the mitochondrial RNA fusion transcript described herein. In one embodiment, at least one of the primers has a nucleotide sequence adapted to hybridize to a junction point or fusion junction in the mtDNA or fusion transcript. In another embodiment, at least one of the primers has a nucleotide sequence adapted to hybridize to a sequence of the mtDNA or fusion transcript adjacent to a junction point or fusion junction in the mtDNA or fusion transcript. In one embodiment, the kit may comprise one or more primers or primer pairs adapted to hybridize to one or more control sequences.

[0113] <(5.0) Exemplary mtDNA Mutations, Fusion Transcripts, Translation Products, Probes, and Primers> Described below are mtDNA mutations (or aberrant mtDNA) and fusion transcripts found to be useful in the methods claimed herein. Predicted translation products are also provided and are believed to be useful for the same reasons. Probe and primer sequences useful for detecting mtDNA and fusion transcripts in a subject are also provided below.

[0114] (5.1) Exemplary mtDNA Mutations Table 1 lists the abnormal mtDNA molecules (i.e., mtDNA molecules having deletions) that were studied. The sequences listed are based on modifications of the wild-type mitochondrial genome (SEQ ID NO: 1) and have been assigned the fusion or "FUS" designation. Where provided, "AltMet" refers to an alternative translation start site. The sequences listed in Table 1 are sections of the mtDNA genome that are recombined or recircularized after removing the subject deletion.

[0115] [Table 1] TIFF0007649745000002.tif142170

[0116] In Table 1, "Deletion ID" is a reference number for identifying the mtDNA deletion among those screened. "SEQ ID NO" refers to the nucleotide sequence identifier assigned to the mtDNA deletion of the subject herein. "Deletion Name" identifies the "FUS" designation, and A:B represents the junction point between the last mitochondrial nucleotide of the first splicing gene and the first mitochondrial nucleotide of the second splicing gene. "Deletion Location" identifies the portion of the respective sequence that was removed from the parent mtDNA molecule. In the next column, "Splice Gene" identifies the spliced ​​gene resulting from the deletion. In this regard, ATP8 represents ATPase8, ATP6 represents ATPase6, CO2 represents COII, and CO1 represents COI. "mtDNA Location" identifies the segment of the mtDNA sequence that corresponds to the wild-type mtDNA genome (i.e., SEQ ID NO:1). "Junction Site" identifies the location of the junction point of the mutant mtDNA following removal of the deletion (based on the wild-type mtDNA genome, SEQ ID NO:1). Thus, by way of example, in the case of deletion ID number 4 (SEQ ID NO: 3), which has "junction site" 7586-7992 / 15730-15887, the deleted mtDNA segment includes nucleotides 7993 through 15729. In such a case, the aberrant mtDNA, once recircularized, contains junctions at nucleotides 7992 and 15730. The portion in brackets in this column identifies the location of the splice in each SEQ ID NO. The final column identifies the flanking repeat sequences of the deletion. The repeats indicated in square brackets are deleted with the deletion indicated in the third column.

[0117] As shown in Table 1, one of the flanking repeat sequences is removed along with the deletion sequence, thus forming part of the deletion sequence. However, the other of the repeat sequences may be included along with the deletion instead. This deletion mechanism is illustrated in FIG. 20, which shows a parent mtDNA molecule 10, where 12 and 20 represent the opposite ends of the mtDNA molecule, and 16 represents the deletion, or the deletion sequence (as listed in the third column of Table 1). The repeat sequences are represented by 14 and 18. During the deletion event, one of the repeats 14 or 18 is removed along with the deletion 16, thus forming part of the deletion 16. Thus, the remaining parent mtDNA, once recircularized, contains the segment 12-18-20 or the segment 12-14-10, as shown in FIG. 20. As described above, although one entire repeat is described as being included in the deletion sequence, only a portion of one or both of the repeats may be included in the deletion.

[0118] A mutant mtDNA sequence according to the present description can include any modification that results in the production of a fusion transcript, non-limiting examples of such modifications include insertions, translocations, deletions, duplications, recombinations, rearrangements, or combinations thereof.

[0119] The step of detecting mtDNA mutations described herein can be selected from any technique known to those skilled in the art.For example, the analysis of mtDNA can include target selection by branching DNA, sequencing of mtDNA, amplification of mtDNA by PCR, Southern, Northern, Western, Southwestern blot hybridization, denaturing HPLC, hybridization to microarray, biochip or gene chip, molecular marker analysis, biosensor, melting temperature profiling, or any combination of the above.

[0120] Variants or fragments of the mtDNA sequences identified herein are also contemplated, and the present description encompasses the use of variants or fragments of these sequences to diagnose and / or monitor endometriosis.

[0121] (5.2) Exemplary Fusion Transcripts Exemplary fusion transcripts for use in the methods described herein are provided in Table 2. These fusion transcripts have been detected and found to be useful in the detection, diagnosis and / or monitoring of endometriosis, as shown in the Examples.

[0122] [Table 2]

[0123] In Table 2, "Transcript No." is an identification number assigned to the fusion transcript and corresponds to the mtDNA deletion ID number in Table 1. "mtDNA Deletion SEQ ID NO." is the mtDNA deletion sequence identifier in Table 1. "Transcript SEQ ID NO." is the sequence identifier of the subject's fusion transcript. "Fusion Transcript Name" indicates the "FUS" designation, with A:B representing the junction point between the last mitochondrial nucleotide of the first spliced ​​gene and the first mitochondrial nucleotide of the second spliced ​​gene. "Flanking Genes" indicates the spliced ​​genes resulting from the deletion. "Deletion Junction" indicates the location of the junction point on the mtDNA molecule following removal of the deletion.

[0124] Naturally occurring fusion transcripts can be extracted from biological samples and identified according to any suitable method known in the art, such as the methods described in the Examples of this Description.

[0125] Fusion transcripts can also be produced by recombinant techniques known in the art, which generally involve transformation (including transfection, transduction, or infection) of a suitable host cell with an expression vector containing the mtDNA sequence of interest.

[0126] Variants or fragments of the fusion transcripts identified herein are also contemplated.

[0127] (5.3) Exemplary translation products of fusion transcripts The deduced amino acid sequences corresponding to the transcripts of mtDNA deletions 1, 4, 14, 16, 120, 122, 193, 400, 516, 586, 8590, and 2767 are provided in Table 3.

[0128] [Table 3]

[0129] [Example] The following examples are provided to further illustrate aspects of the present invention and are not intended to limit the scope of the specification in any way.

[0130] Example 1: Large-scale fusion transcript screening in endometrial tissue 268 probes corresponding to the fusion transcripts were screened with endometrial tissue samples for evidence of differential expression in samples obtained from endometriosis patients versus control samples. Screening methods and results are described hereinafter.

[0131] Probe library generation The 268 probes were identified using a proprietary program that finds nucleotide base pair repeats. This program identified over 16,000 possible deletions based on direct and indirect repeat elements flanking the sequence to be deleted at the 5' and 3' ends. The selection of the 268 probes was based on the criteria that a minimum of 8 base pair repeats was required; however, deletions with fewer than 8 base pair repeats are possible. As an example, the repeat of deletion 16 is 3 bp.

[0132] Tissue samples Large endometrial samples (>0.49 g) were obtained. The tissue "status" or diagnosis, as determined by the physician at the time of surgery, and the cause(s) of surgery are shown in Table 4.

[0133] [Table 4]

[0134] Tissue homogenates were prepared using the QuantiGene™ Sample Processing Kit for “Fresh or Frozen Animal Tissue” using the samples listed in Table 4. For each sample, four pieces of frozen endometrial tissue were cut and weighed (approximately 100 mg each) before being added to 6 mL of disruption solution containing 60 μL of proteinase K. Samples were homogenized using Qiagen's tissue disruption probe and incubated overnight at 65° C. The homogenates were then clarified by two centrifugations at 16000×g for 15 min. The supernatant was saved and used as template for the following branched DNA assay. Alternatively, DNA was extracted from tissue homogenates or directly from fresh frozen tissues according to the tissue protocol using Qiagen's QiaAmp™ DNA Mini Kit. DNA was then quantified with a Nanodrop™ spectrophotometer and standardized for subsequent use in qPCR reactions.

[0135] Mitochondrial DNA deletions and the resulting fusion transcripts can be detected using one of a number of molecular techniques. Here, branched DNA and quantitative PCR techniques were used for the detection of fusion transcripts and parental aberrant mtDNA molecules, respectively.

[0136] Branched DNA Platform For tissue samples, Panomics' Quantigene™ 2.0 protocol for "Capturing Target RNA from Fresh, Frozen, or FFPE Tissue Homogenates" was followed. A working probe set consisting of water, lysis solution, blocking reagent, and probe was first added to the capture plate. The probes (or "capture probes") used in this example comprise oligonucleotides (having complementary nucleotide sequences) designed to bind to the junction points of mtDNA encoding each of the fusion transcripts listed in Table 2. In particular, the probes used for branched DNA (bDNA) analysis were the probes listed in Table 5 below.

[0137] The homogenate was then added to the capture plate and incubated overnight at 55°C to allow for probe-template hybridization. Following a series of washes and hybridization steps, a chemiluminescent substrate was added. Decomposition of alkaline phosphatase bound to the probe-template hybrid results in a luminescent signal reported as relative luminescence units (RLU). Each capture plate was read in duplicate and the RLU was measured with a Promega Glomax™ luminometer. The RLU values ​​were bioinformatically analyzed. The two plate reads and the triplicate values ​​were averaged, respectively, provided that they had a CV (coefficient of variation; i.e., ratio of standard deviation to mean) of 15%. It was also determined whether the RLU value was above background for a given probe. Specifically, the lower limit of quantification, LOQ (LOQ=probe background RLU mean+10 standard deviation of background mean), was calculated and subtracted from the sample RLU. The sample RLU values ​​were then converted to log2 or log10 values ​​for ease of analysis. Finally, for any given probe, sample RLU was normalized to housekeeper (HK) RLU by subtracting or dividing the sample RLU from the sample RLU. The normalized results testing probes 1, 4, 14, 16, 120, 122, 193, 400, 516 and 586 (where the probe numbers correspond to the fusion transcript numbers given above) with three endometriosis positive and four control endometrial samples are shown in Figures 2A-2J and summarized in Table 5 below.

[0138] [Table 5]

[0139] Table 5 shows the normalized mean RLU values ​​(Log2LOQProbe-Log2LOQHK23) for control and endometriosis-positive ("Endo.Pos.") tissue samples (corresponding to the scatter plots shown in Figures 2A-2J). The mean difference and the significance of the difference between the two tissue groups are given. The mean copy numbers of a given fusion transcript (i.e., probes 1, 4, 14, 16, 400, 586, 120, 122, 193 and 516) are also shown in Table 5.

[0140] qPCR reaction qPCR analysis was performed for transcript numbers 1, 4, 14, 16, 120, 122, 193, 586, 8590, and 2767 (see Table 6 below). Purified DNA extracts were normalized to a concentration of 0.25ng / μL using nuclease-free ultrapure water. qPCR reactions were performed at room temperature under dim light using Qiagen's Quantitect™ Sybr Green® PCR kit. 10 μL of template was added to 12.5 μL of 2X master mix along with 0.025-0.0625 μL each of 100 μM forward and reverse primers depending on the target. Primer sequences were designed for specific DNA targets and these sequences are shown in Tables 6 (junction primers) and 7 (flanking primers). As used herein, the term "junction primer" is understood to mean a primer that hybridizes to a region of a target DNA molecule that has at least one of the nucleotide pairs that form a junction point after removal of the deletion. Thus, in one embodiment, the junction primer can overlap both nucleotides that form the junction point, or only one of the nucleotides. In some cases, more than one pair of primers was used, as shown in the table below. The reaction was brought to a final volume of 25 μL with PCR grade H2O. The reaction mixture was cycled in either a Chromo 4™ (Biorad) or Opticon 2™ (MJ Research) real-time PCR cycler.

[0141] [Table 6] TIFF0007649745000008.tif218170TIFF0007649745000009.tif231170TIFF0007649745000010.tif169170

[0142] [Table 7] TIFF0007649745000012.tif121170

[0143] Results and Discussion As shown above, about 268 fusion transcripts were screened in the course of this study, and from this study, 10 endometriosis markers were selected for further study, as discussed in more detail herein.In particular, as described herein, the elevated levels of fusion transcripts associated with deletion ID numbers 1, 4, 14, 16, 120, 122, 193, 400, 516 and 586 (i.e., transcripts of SEQ ID NOs: 13-15 and 17-23, respectively) in endometrial tissue were found to be associated with endometriosis.The presence of each transcript was determined by assaying for each probe that has at least a complementary nucleotide sequence to the portion of the transcript that has the junction point.

[0144] Scatter plots and performance of all fusion transcript probes are shown in Figures 2A-2J and Table 5. Figure 3 shows the location of the fusion transcripts throughout the mitochondrial genome, the gene location within the genome, and the location of the 10 mtDNA fusion transcripts (i.e., "probes" or "targets") of the present invention within the genome (indicated by lines spanning the length of each deletion). Probes corresponding to the aforementioned fusion transcripts were tested on three endometriosis-positive and four endometriosis-negative endometrial samples (see Table 1). For each sample, RLU values ​​were normalized against the RLU values ​​obtained for the housekeeping gene transcripts HK23 (Human Beta-2-microglobulin), HK25 (Human GAPD), and HK18 (Peptidyl-prolyl isomerase B).

[0145] Based on the results of this study, it is concluded that fusion transcripts 1, 4, 14, 16, 120, 122, 193, 400, 516, and 586 (i.e., transcripts having sequences shown in SEQ ID NOs: 13-15 and 17-23, respectively) can be used in the detection of endometriosis, particularly by assaying endometrial tissue. In particular, this study found that elevated levels of the transcripts of interest in endometrial tissue samples are highly correlated with endometriosis. Detection of the fusion transcripts of interest can be achieved using the probes identified above, which have a nucleotide sequence at least sufficiently complementary to the nucleotide sequence of at least a portion of each fusion transcript, the portion including a junction point such that the probe hybridizes to each fusion transcript.

[0146] Based on these findings, it is also concluded that high levels of abnormal mtDNA having the identified or greater deletions 1, 4, 14, 16, 120, 122, 193, 400, 516, and 586 (i.e., deletions having the nucleotide sequences shown in SEQ ID NOs: 2-4 and 6-12, respectively) can be used to detect endometriosis. Such deletions can be identified by identifying junction points in the parent mtDNA (i.e., the recircularized major subliminal) after recircularization. Junction points can be identified using a probe having a nucleotide sequence that is at least substantially complementary (such that the probe hybridizes to the respective mtDNA) to at least a portion of the mtDNA nucleotide sequence that contains the junction point. Junction points can also be identified using primers, at least one of which has a nucleotide sequence that is substantially complementary to the mtDNA nucleotide sequence that contains the junction point. Alternatively, the primers can include a pair having a nucleotide sequence that is at least substantially complementary to the mtDNA sequence adjacent to the junction point.

[0147] Similarly, it can be concluded that deletions can also be identified by identifying the junction points of the deleted sequence (i.e., the recircularized subunit) after recircularization.

[0148] Translation products from fusion transcription products (ie, fusion proteins having the amino acid sequences shown in SEQ ID NOs: 24 to 26, 28 to 34, and 84, respectively) may also be usable in such detection methods.

[0149] Thus, as described herein, there is provided a method for the detection of endometriosis, which comprises the use of probes and primers for the identification of said fusion transcripts or abnormal mtDNA. These probes and primers have nucleic acid sequences complementary to said mitochondrial fusion transcripts and their abnormal parent mtDNA molecules, respectively. In particular, the probes described herein are designed to be at least substantially complementary to the fusion transcripts that code for the transcribed junction points corresponding to the recombined (or recircularized) mtDNA. The primers described herein are preferably designed such that one of the primer pairs has a nucleotide sequence complementary to the junction point of the abnormal mtDNA that has been recircularized following the removal of the deletion as described herein. It will also be understood that other primer pairs can be designed, where one of the primer pairs is at least substantially complementary to the junction point of the deleted sequence that has been recircularized, or where the primer pair is at least substantially complementary to the mtDNA nucleotide sequence adjacent to the junction point.

[0150] Example 2: Detection of mtDNA deletions in circulating blood samples In this example, mitochondrial DNA, mtDNA, deletions were investigated as potential biomarkers for endometriosis. The study focused primarily on mtDNA deletions obtained from circulating blood samples. Seven deletions were investigated. Two of these deletions, the 1.2 kb deletion and the 3.7 kb deletion, discussed further below, were determined to have high diagnostic accuracy as biomarkers using minimally invasive blood samples taken from fertile women with symptoms of endometriosis. The 1.2 kb and 3.7 kb deletions have been discussed above, with the 1.2 kb deletion identified as deletion "193" and the 3.7 kb deletion identified as deletion "14" or "14a." The characteristics of these deletions have been summarized previously in Table 1 and are presented again in Table 8. References to the 3.7 kb deletion herein refer to deletion 14 or deletion 14a.

[0151] [Table 8]

[0152] As mentioned above, the 1.2 kb deletion refers to the deletion of nucleotides 9087-10312 based on the wild-type mtDNA genome (SEQ ID NO: 1). Such a deletion thus results in a major sublimon having bases 0-9086 and 10313-16568 (which, when recircularized, has a junction between nucleotides 9086 and 10313). Similarly, the 3.7 kb deletion refers to the deletion of nucleotides 9189-12905, resulting in a major sublimon having bases 0-9188 and 12906-16568 (which, when recircularized, has a junction between nucleotides 9188 and 12906).

[0153] As mentioned above, recircularization of the major sublimon is discussed, but recircularization of such minor sublimons is also appropriate if the minor sublimon being recircularized has a unique junction point as taught. In this study, minor sublimons corresponding to the 1.2 kb and 3.7 kb deletions were identified during the process of sequencing the samples. Thus, the findings in this example can be extended to the detection of minor sublimons resulting from the deletions described herein.

[0154] method Participants and sample collection This study utilised unprocessed, de-identified clinical samples collected from patients prospectively enrolled as part of the EndOx study at the Oxford Endometriosis Medical Centre, John Radcliffe Hospital, University of Oxford. Briefly, samples were collected from women scheduled to undergo laparoscopy for suspected endometriosis because of pelvic pain (symptomatic) or tubal ligation (asymptomatic). Study participants were women aged 18 years or older (premenopausal) and confirmed as non-pregnant. All samples were obtained under a study protocol that received appropriate ethics committee approval (Oxfordshire REC A, 09 / H0604 / 58) from the National Research Ethics Service. All clinical samples were anonymised to protect the identity of the patients from whom they were obtained. The study was designed, conducted and reported in accordance with the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICPR) “Harmonised Trilateral Guidelines on Good Clinical Practice for Medicinal Products for Human Use” and in accordance with applicable national regulations and the ethical principles set out in the Declaration of Helsinki. All patients gave written informed consent prior to participation.

[0155] Blood specimens and extensive clinical and phenotypic data were collected before surgery. Study samples were collected, transported, and stored according to standardized WERF EPHect procedures [26, 41-44].

[0156] Patient population / study cohort Clinical specimens used in this study were classified as asymptomatic controls, symptomatic controls without surgically confirmed endometriosis, or cases with surgically confirmed endometriosis. Asymptomatic controls were defined as specimens obtained from patients undergoing scheduled tubal ligation who were not clinically suspected of endometriosis and were surgically confirmed to be free of endometriosis. Symptomatic controls were defined as specimens obtained from patients with pain or other symptoms (except infertility) who were clinically suspected of endometriosis but had no endometriotic lesions visualized by laparoscopy by an experienced gynecologic surgeon.

[0157] Endometriosis was scored by the operating surgeon using the revised American Society for Reproductive Medicine (rASRM) classification of endometriosis.[ 45 ] Cases were classified by disease subtype (peritoneal, ovarian, or deep endometriosis) and rASRM stage (stages I to IV, representing minimal, mild, moderate, and severe disease).

[0158] Sample handling, processing, and mtDNA amplification DNA extraction Total DNA was extracted from 200 μL of plasma using a QIAamp 96 QIAcubeHT™ extraction kit (Qiagen, Crawley, UK) automated on a QIAcube HT™ system (Qiagen, Crawley, UK). Extracted DNA was eluted in 200 μL of AE buffer.

[0159] Real-time qPCR for mtDNA deletions Amplification was carried out in 20 μL reactions using 96-well microplates (Bio-Rad, Hemel Hempstead, UK). Each well contained 5 μL of non-standardized DNA template, 1×SYBR Green® master mix, and 250 nM of each primer. The primers used in the reactions are shown in Table 9.

[0160] [Table 9]

[0161] PCR and SYBR Green® I fluorescence were analyzed using the Chromo 4™ Real-time PCR Detection System (Bio-Rad, Hemel Hempstead, UK). Cycle conditions for the 1.2 kb deletion were as follows: 95°C for 3 min, followed by 5 cycles of 95°C for 30 s, 67°C for 30 s, and 72°C for 30 s; the annealing temperature was decreased by 0.5°C for each subsequent cycle. Amplification conditions were 45 cycles of 95°C for 30 s, 65°C for 30 s, and 72°C for 30 s. All other deletions were amplified with the standard protocol of 45 cycles of 95°C for 30 s, 58-65°C for 30 s, and 72°C for 30 s. Amplification was followed by a 70°C-90°C melting curve analysis with readings every 0.5°C. Each plate of samples and controls was amplified in triplicate in triplicate.

[0162] Standardization of Real-time qPCR with 18S rRNA Target amplicon amounts were normalized using the nuclear DNA gene for 18S rRNA. Amplification reactions were performed in 20 μL reactions in 96-well microplates. Each well contained 5 μL of non-normalized DNA template, 1× SYBR Green® master mix, and 200 nM of each primer. Amplification and SYBR Green® I fluorescence were analyzed using a Chromo4 real-time PCR detection system. Amplification conditions were 95°C for 3 min, followed by 40 cycles of 95°C for 30 s, 64.5°C for 30 s, and 72°C for 30 s. Amplification was followed by a 70°C to 90°C melting curve analysis with readings every 0.5°C.

[0163] quality control Quantitation cycles (Cq) were calculated using a regression model in CFX Manager software (Bio-Rad, Hemel Hempstead, UK). The Cq of each deletion amplicon was normalized to the Cq of the other copy nuclear target 18s rRNA gene amplicon. All samples were amplified in triplicate on separate plates and were considered eligible if at least two of the three replicates were within 1.5 Cq and had melting temperatures (Tm) consistent with the target amplicon when present (Tm 81°C ± 2°C for deletions, Tm 82°C ± 2°C for 18S rRNA).

[0164] Two no-template control samples were run alongside each batch of DNA extraction and were confirmed negative for amplification of both the deletion target and the 18S rRNA gene. Two no-template control reactions were included with each PCR plate and were confirmed negative for amplification of both the deletion target and the 18s rRNA gene. The specificity of the deletion primers was assessed using rho 0 cellular DNA (to detect mitochondrial pseudogene amplification) as well as DNA from a buccal swab of a healthy male and DNA extracted from the rho 0 parental cell line (prior to mitochondrial deletion).

[0165] For the first round of standard PCR reactions, DNA extracted from patients with confirmed endometriosis underwent whole genome amplification using the Repli-G™ Mitochondrial DNA Kit (Qiagen) to ensure sufficient DNA quantities during this step.

[0166] Preparation of Rho 0 cells Rho 0 cells were prepared as previously described

[46] . Briefly, cells from the human osteochalcoma cell line 143B (ATCC CRL-8303) were treated with ethidium bromide to deplete cytoplasmic mitochondrial DNA. Cells were grown to confluence in high glucose DMEM with pyruvate, L-glutamine, uridine (50 μg / ml) and 5% FBS.

[0167] statistical analysis No formal sample size calculation was performed, and the number of clinical specimens used was deemed sufficient to meet the study objectives.

[0168] For qPCR, targets were amplified in triplicate from all specimens and average Cq values ​​were calculated. We determined standardized deletion values ​​(ΔCq) by quantifying the deletion amplicon relative to the 18S rRNA reference amplicon. Statistical analysis was performed using Graphpad Prism™ 5.0 (Graphpad software Inc, La Jolla, CA, USA) for receiver operating characteristic (ROC) curves and descriptive statistics. Correlation and significance tests were performed using SPSS v17.0 (IBM Corp, Armonk, NY, USA). We summarized clinical characteristics using counts and percentages for categorical data and means, standard deviations (SD), and ranges for continuous variables. The means of the two groups were compared using Student's t-test and Mann-Whitney U-test for parametric and nonparametric distributions, respectively. Correlation between two variables was evaluated with the Pearson correlation coefficient (r). For the presence of endometriosis, ROC curves were generated for all but the 6.5 kb deletion. The area under the curve (AUC) of the ROC, as well as the sensitivity and specificity at selected cutoffs (described below) were calculated with 95% confidence intervals (CI). For all tests, a p-value <0.05 was considered statistically significant.

[0169] result Patient population and clinical specimens Demographic and clinical characteristics for the patients who provided the clinical samples used for evaluation of the 1.2 kb and 3.7 kb deletions are summarized in Table 10.

[0170] [Table 10]

[0171] Abbreviations: N = number of patients / sample; SD = standard deviation. (1) Means (standard deviations) are shown; for patients whose age at the time of sample collection was available, the mean and standard deviation were calculated. (2) Patients' status within 3 months of sample collection. (3) Menstrual status of patients at the time of sample collection.

[0172] The clinical and demographic characteristics of the patients and specimens used in the evaluation of the 1.2 kb and 3.7 kb were similar and differed as a result of including only those samples with 18S rRNA and paired qPCR results for each deletion that met the acceptance criteria described above.

[0173] 1.2 kb deletion cohort One hundred and seventy-one samples were used for evaluation of the 1.2 kb deletion. The mean (SD) age of patients providing samples was 34.2 (6.8) years. The mean age was similar for the control and case groups with mean (SD) ages of 36.6 (6.9) and 33.7 (6.7) years, respectively, and the difference was not statistically significant (p=0.113). Of the 171 patient samples used for evaluation, 116 (67.8%) patients reported no hormonal therapy within 3 months prior to sample collection, 48 (28.1%) reported hormonal therapy within 3 months prior to sample collection, and 7 (4.1%) reported undetermined. Menstrual cycle phase data were calculated using the last menstrual period (LMP) prior to the date of blood collection in relation to the patient's normal cycle length. Twenty-four (14.0%) patients reported not having a menstrual period - 19 of them were on hormone therapy, 12 (7.0%) were irregular menses, 31 (18.1%) were in the menstrual phase (between 1 and 5 days after the first day of LMP), 44 (25.7%) were in the follicular phase (5 to 14 days after LMP), and 60 (35.1%) were in the luteal phase plus extended menstrual phase (>15 days after LMP).

[0174] The control group included a total of 28 specimens; 18 (64.3%) from symptomatic patients (with symptoms consistent with endometriosis other than infertility and confirmed absence of surgery for the disease) and 10 (35.7%) from asymptomatic patients scheduled for tubal ligation. The test group included 143 specimens from patients with three disease subtypes (peritoneal, ovarian, and deep infiltrating [DI] endometriosis) classified into four stages (rASRM I–IV). Forty-nine specimens (34.3%) were from women with peritoneal endometriosis, 45 (31.5%) from women with ovarian endometriosis, and 49 (34.3%) from women with deep endometriosis. Sixty-three specimens (44.1%) were from patients with stage I disease, 21 (14.7%) were stage II, 29 (20.3%) were stage III, and 28 (18.6%) were stage IV. Two specimens (1.4%) were of unknown disease stage.

[0175] 3.7 kb deletion cohort One hundred eighty-one samples were used for the 3.7 kb deletion evaluation. The mean (SD) age of patients who provided samples was 34.4 (6.9) years. The mean age was similar in the control and case groups, with mean (SD) ages of 37.2 (6.8) and 33.8 (6.8), respectively, and the difference was not statistically significant (p=0.166). One hundred nineteen (65.7%) patients reported no hormonal therapy within 3 months prior to sample collection, 55 (30.4%) had received hormonal therapy within 3 months prior to sample collection, and 7 (3.9%) were uncertain. Twenty-six (14.4%) patients reported no menses, 15 (8.3%) reported irregular menses, 31 (17.1%) reported menstrual phase (1-5 days), 44 (24.3%) reported follicular phase (5-14 days), and 65 (35.9%) reported luteal phase + extended menstrual phase (≥15 days).

[0176] The control group included 32 specimens: 19 (58.4%) from symptomatic patients and 13 (40.6%) from asymptomatic patients. The test group included 149 specimens: 52 (34.9%) from women with peritoneal endometriosis, 47 (31.5%) from women with ovarian endometriosis, and 50 (33.6%) from women with deep endometriosis. 65 specimens (43.6%) were from women with stage I disease, 24 (16.1%) from stage II, 30 (20.1%) from stage III, and 28 (18.8%) from stage IV. Two specimens (1.3%) were of unknown disease stage.

[0177] mtDNA deletions and preliminary evaluation – standard PCR Seven candidate deletions were initially selected for evaluation based on sequence composition, the presence of adjacent repeated positions within the major arc of the mitochondrial genome where a relatively large number of deletions have been reported

[47] , and previous observations to date in endometrial tissue (data not shown). Deletions were selected within the following genomic regions: CO2 to ATP6 (1.0 kb deletion); ATP6 to ND3 (1.2 kb deletion); ATP8 to ND4 (2.4 kb deletion); ATP6 to ND5 (3.7 kb deletion); ATP8 to ND5 (5.0 kb deletion); CO1 to ND5 (6.5 kb deletion); and CO2 to CytB (7.7 kb deletion). An initial round of standard (quantitative) PCR and visualization after gel electrophoresis was used to prequalify each of the deletion targets and to determine whether each of the candidates was (i) detectable; (ii) in sufficient copy number for reliable detection; (iii) had the expected amplicon size; and (iv) specific and did not co-amplify with nuclear pseudogenes or generate non-specific amplification products.

[0178] All seven predicted deletions were detectable in circulating plasma. However, the 5.0kb and 6.5kb deletions amplified rho 0 cell DNA, indicating potential coamplification of nuclear mitochondrial pseudogenes (numts). Furthermore, the 6.5kb deletion was insufficiently copy number and was not considered a viable candidate for further QPCR testing. The 7.7kb and 2.4kb deletions were in low copy number but still detectable by QPCR methods and were therefore subjected to further evaluation. The 5.0kb deletion amplified DNA from a buccal swab of a healthy male, indicating a potential lack of disease specificity. The 7.7kb deletion also showed low levels of amplification from this sample.

[0179] The remaining six deletions were further evaluated using QPCR to determine whether the targets were i) in sufficient copy number without whole genome amplification, ii) with sufficient diagnostic accuracy, iii) detectable in rho 0 cells using more sensitive QPCR, and iv) the precision of the assay was acceptable. Acceptable precision criteria were a maximum deviation of 1.5 Ct between a minimum of two of three replicates for each of the targeted deletions.

[0180] Preliminary evaluation using clinical samples As a preliminary evaluation of the remaining six candidates, the deletions were evaluated using a set of 55 clinical specimens; 46 from patients with confirmed endometriosis and 9 from symptomatic control patients. After initial QPCR testing, the 2.4 kb deletion was determined to have insufficient copy number, the 1.0 kb deletion amplified DNA extracted from rho 0 cells (indicating coamplification of numts), and the 7.7 kb deletion amplified only at low stringency annealing temperatures (implying mispriming events are more likely). Although these candidate deletions did not meet the requirements of the assay as planned here, they may still stand as biomarkers that would benefit from further assay optimization to obtain better sequence specificity and assay sensitivity.

[0181] Of the seven deletions initially selected, the 1.2 kb and 3.7 kb deletions were present in plasma in sufficient copy numbers to facilitate reliable and easy detection. The assay was specific under the PCR conditions tested and accurately distinguished between healthy (asymptomatic) control specimens and specimens from confirmed endometriosis patients (data not shown). Furthermore, both the 1.2 kb and 3.7 kb deletions were accurate in distinguishing between symptomatic controls and endometriotic disease cases (all subtypes and stages combined). The AUC (95% CI) for the 1.2 kb deletion was 0.8116 (0.6178 to 1.005), which was statistically significant (p = 0.0034). Similarly, the AUC (95% CI) for the 3.7 kb deletion was 0.8478 (0.6663 to 1.029), which was also significant (p = 0.0011; Table 11).

[0182] [Table 11]

[0183] Abbreviations: AUC = area under the curve; CI = confidence interval; N = number of samples in the evaluation set; PCR = polymerase chain reaction.

[0184] Diagnostic accuracy of 1.2-kb and 3.7-kb deletions To more fully evaluate the 1.2kb and 3.7kb deletions as clinically actionable biomarkers of endometriosis, the ability of these deletions to discriminate between symptomatic controls and all types of endometriosis combined, between the three subtypes of endometriosis, and between the four stages of endometriosis was determined using a larger population of clinical specimens (Table 10). Valid paired results (both targeted and 18S gene amplification) were obtained for 171 specimens with the 1.2kb deletion and 181 specimens with the 3.7kb deletion. These analyses were performed using only symptomatic controls and confirmed disease specimens to more accurately reflect the clinically relevant patient population (i.e., women exhibiting symptoms of endometriosis with surgical confirmation of disease status as an outcome). Importantly, the 1.2kb and 3.7kb deletions did not detect differences between symptomatic and asymptomatic control specimens at p=0.462 and p=0.878, respectively.

[0185] Symptomatic control vs. total disease Similar to preliminary analysis of 55 clinical samples, both the 1.2 kb and 3.7 kb deletions accurately distinguished between symptomatic control samples and endometriotic disease samples (peritoneal, ovarian, and deep endometriosis samples combined). The AUC (95% CI) for the 1.2 kb deletion was 0.7879 (0.6791-0.8967), which was statistically significant (p<0.0001). The AUC (95% CI) for the 3.7 kb deletion was 0.807 (0.7063-0.9077), which was also significant (p<0.0001; Figures 4A and 4B). Receiver operating characteristic (ROC) curve coordinates were examined and thresholds, or cutoffs, were selected to optimize sensitivity. Applying a threshold of -4.43 to distinguish between symptomatic controls and all subtypes / stages of endometriosis using the 1.2 kb deletion yielded sensitivity and specificity values ​​of 81.8% and 72.2%, respectively. At a threshold of 10.51, the sensitivity for the 3.7 kb deletion was 85.1% and the specificity was 57.9% (Table 12).

[0186] [Table 12]

[0187] Combining the 1.2-kb and 3.7-kb deletions improved diagnostic accuracy between all symptomatic controls and all endometriosis (AUC 0.827 (0.722-0.931) and between symptomatic controls and stage I / II disease (AUC 0.882 (0.784-0.980)) (data not shown).

[0188] Disease by subtype - 1.2 kb deletion An important feature of any diagnostic aid for endometriosis is the ability to accurately detect all disease subtypes. We evaluated the ability of the 1.2 kb deletion to discriminate between symptomatic control specimens and specimens from patients with confirmed peritoneal, ovarian, and deep endometriosis. The distribution of the 1.2 kb deletion for each disease subtype is shown in Figure 5A. The mean (SD) ΔCt values ​​were -4.312 (2.075) for symptomatic controls, -7.187 (2.581) for peritoneal disease, -6.291 (2.344) for ovarian disease, and -6.193 (2.143) for deep endometriosis. The difference in the normalized amount of the 1.2 kb deletion between symptomatic controls was statistically significant for peritoneal (p<0.0001), ovarian (p=0.003), and deep endometriosis (p=0.0012).

[0189] The diagnostic accuracy of the 1.2 kb deletion is shown in the figure (AUC (95% CI) values ​​are 0.8549 (0.7425-0.9672), p<0.0001 for the detection of peritoneal endometriosis, 0.7457 (0.6118-0.8796), p=0.0025 for the detection of ovarian endometriosis, and 0.7596 (0.6292-0.8901), p=0.0012 for deep endometriosis). Taken together, these data show that the 1.2 kb deletion allowed accurate discrimination of samples taken from symptomatic controls and patients with peritoneal, ovarian and deep endometriosis. Applying a threshold of -4.430 for the discrimination of symptomatic controls and peritoneal endometriosis tp using the 1.2 kb deletion, the sensitivity and specificity values ​​are 81.8% and 72.2%, respectively. At a threshold of -4.675, the sensitivity and specificity of the 1.2 kb deletion in distinguishing between symptomatic controls and ovarian endometriosis are 75.6% and 72.2%, respectively. At a threshold of -4.350, the sensitivity and specificity of the 1.2 kb deletion in distinguishing between symptomatic controls and deep endometriosis are 78.6% and 66.7%, respectively (Table 12).

[0190] Disease by subtype - 3.7 kb deletion The distribution of the 3.7 kb deletion for each disease subtype is shown in Figure 6A. The mean (SD) ΔCt values ​​were 11.12 (2.239) for symptomatic controls, 7.569 (1.843) for peritoneal endometriosis, 8.549 (2.089) for ovarian endometriosis, and 8.617 (2.125) for deep endometriosis. The difference in amplicon abundance between symptomatic controls was statistically significant for peritoneal endometriosis (p<0.0001), ovarian endometriosis (p<0.0001), and deep endometriosis (p=0.0072). The diagnostic accuracy of the 3.7 kb deletion to detect each of the three disease subtypes is shown in Figures 6B-6D (AUC (95% CI) values ​​are 0.8978 (0.8131-0.9824), p<0.0001 for detecting peritoneal endometriosis, 0.8158 (0.7003-0.9313), p<0.0001 for detecting ovarian endometriosis, and 0.7110 (0.5746-0.8475), p=0.0071 for detecting deep endometriosis). Taken together, these data show that the 3.7 kb deletion significantly improved the diagnostic accuracy of the 3.7 kb deletion in specimens taken from symptomatic controls and women with peritoneal, ovarian and deep endometriosis. It shows that the 3.7 kb deletion could accurately distinguish between symptomatic controls and peritoneal endometriosis. Applying a threshold of 8.805 for the differentiation of symptomatic controls and peritoneal endometriosis with the 3.7 kb deletion gives sensitivity and specificity values ​​of 88.5% and 73.7%, respectively. At a threshold of 8.910, the sensitivity and specificity of the 3.7 kb deletion in differentiating between symptomatic controls and ovarian endometriosis is 80.9% and 68.4%, respectively. At a threshold of 11.01, the sensitivity and specificity of the 3.7 kb deletion in differentiating between symptomatic controls and deep endometriosis is 80.0% and 52.6%, respectively (Table 12).

[0191] Disease by stage - 1.2 kb deletion Another important feature of a biomarker for endometriosis is its ability to detect both low and high disease stages. Next, the ability of the 1.2 kb deletion to distinguish between symptomatic control specimens and specimens with confirmed low (I / II) or high (III / IV) disease stages was evaluated. The distribution of the 1.2 kb deletion for stage I / II and stage III / IV disease is shown in Figure 7A. The mean (SD) ΔCt values ​​were -4.312 (2.075) for symptomatic controls, -6.692 (2.505) for stage I / II, and -6.348 (2.25) for stage III / IV. The difference between symptomatic controls was statistically significant for stage I / II (p<0.0001) and stage III / IV (p=0.001) disease groups. The difference between stages I / II and III / IV was not statistically significant (p=0.406).

[0192] The diagnostic accuracy of the 1.2 kb deletion is shown in Figures 7A-7C (AUC (95% CI) values ​​were 0.7989 (0.6868-0.9111), p<0.0001 for detection of stage I / II and 0.7661 (0.6398-0.8924), p=0.0007 for detection of stage III / IV. Thus, the 1.2 kb deletion could accurately distinguish between symptomatic controls and all stages of the disease. At a threshold of -4.430, the sensitivity and specificity of the 1.2 kb deletion in distinguishing between symptomatic controls and stage I / II endometriosis are 82.1% and 72.2%, respectively. At a threshold of -4.490, the sensitivity and specificity of the 1.2 kb deletion in distinguishing between symptomatic controls and stage III / IV endometriosis are 80.7% and 72.2%, respectively (Table 12).

[0193] Disease by stage - 3.7 kb deletion The distribution of the 3.7 kb deletion for stages I / II and III / IV disease is shown in Figure 8A. The mean (SD) ΔCt values ​​were 11.12 (2.239) for symptomatic controls, 8.243 (2.156) for stage I / II, and 8.112 (2.14) for stage III / IV. The difference between symptomatic controls was statistically significant for the stages I / II (p<0.0001) and III / IV (p=0.0008) disease groups. The diagnostic accuracy of the 3.7 kb deletion is shown in Figures 8B-8C (AUC (95% CI) values ​​were 0.8383 (0.7412-0.9353), p<0.0001 for detection of stage I / II and 0.7591 (0.6354-0.8837), p=0.0007 for detection of stage III / IV. The difference between stages I / II and III / IV was statistically significant for the 3.7 kb deletion (p=0.016). These data are consistent with the results for the 3.7 kb deletion. These results show that the 3.7 kb deletion was able to accurately distinguish between symptomatic controls and all stages of the disease. At a threshold of 10.17, the sensitivity and specificity of the 3.7 kb deletion in distinguishing between symptomatic controls and stage I / II endometriosis are 87.6% and 63.2%, respectively. At a threshold of 11.00, the sensitivity and specificity of the 3.7 kb deletion in distinguishing between symptomatic controls and stage III / IV endometriosis are 84.5% and 52.6%, respectively.

[0194] Correlation with patient age, specimen age, hormone therapy and menstrual phase - 1.2 kb deletion An ideal biomarker test would give accurate results independent of patient and sample age, hormone therapy treatment, and timing of the menstrual phase during sample collection. The impact of these parameters on disease detection is summarized in Table 13.

[0195] [Table 13]

[0196] (1) T-test was used to examine the effect of hormonal status on the detection of endometriosis.

[0197] (2) ANOVA was used to examine the effect of menstrual cycle on the detection of endometriosis.

[0198] For the 1.2 kb deletion, we determined that there was no correlation between disease detection and patient age; the correlation coefficient (r) was r=0.030 (p=0.698). Additionally, there was no correlation between disease detection and sample age by year of collection (r=0.072, p=0.353). When stratified by hormonal status (patients receiving or not receiving hormonal therapy within 3 months prior to sample collection), the difference in disease detection was not statistically significant (p=0.120). When stratified by menstrual phase (absent, irregular, menstruating, follicular, luteal + expanded menstrual), there was no statistically significant difference in disease detection with the 1.2 kb deletion (p=0.228).

[0199] Similarly, disease detection based on the 3.7 kb deletion was not significantly correlated with patient age (r=0.1034; p=0.166) or sample age (r=0.0628; p=0.4009), or significantly affected by hormone therapy (p=0.195). Detection of endometriosis by the 3.7 kb deletion was significantly correlated with menstrual phase (p=0.036), with a clear difference in detection between patients not in period and those in the follicular phase (p=0.026), with a difference of 1.72. Taken together, these data indicate that the accuracy of the 1.2 kb and clinically relevant variables is not significantly affected by these variables, and that the accuracy of the 3.7 kb deletion is slightly affected by menstrual phase.

[0200] Consideration Endometriosis is a highly prevalent disease in women of reproductive age, associated with a large economic burden and resulting in a significant decrease in the quality of life of affected women. One of the major contributing factors to this clinical problem is the lack of diagnostic tools to facilitate early detection and intervention. The current diagnostic gold standard is a thorough visual examination by laparoscopy, ideally followed by histological confirmation of suspicious lesions [5,15]. Only a few objective data are available, so the diagnostic value of this method is largely unknown. The use of laparoscopy is considered by some to be potentially inaccurate, even when combined with the accuracy of histological confirmation, which reportedly ranges from 60% to 85% [48-51]. Standard diagnostic methods can be further complicated in cases where the disease itself presents atypically, or in early stages of the disease that are not easily visualized and are overlooked by inexperienced surgeons. Furthermore, if medical intervention can be initiated in an attempt to avoid or delay surgery, a presumptive diagnosis of endometriosis based on accurate biomarker testing would provide the necessary evidence to support this treatment. Thus, there is a clear need to improve on current standards so as to provide more consistent results, especially early in the disease process.

[0201] In the current study, we identified and evaluated two novel mtDNA deletions as potential biomarkers for endometriosis. Assays targeting the 1.2kb and 3.7kb deletions meet the criteria for a sound diagnostic test, utilize minimally invasive specimens, and (if successfully translated into clinical use) could potentially help shorten the time-to-diagnosis delay associated with current diagnostic practice, providing an opportunity for medical intervention before surgery. After establishing diagnostic thresholds (as described above), the sensitivity of the 1.2kb deletion assay was 81.8% and the specificity was 72.2%. The diagnostic performance of the 3.7kb deletion assay was similar, with sensitivity and specificity of 85.1% and 57.9%, respectively. Thus, a diagnostic assay based on either of these deletions could complement the current standard of care. Of particular importance is the diagnostic accuracy of these deletions for early stage disease, since later stage disease is more easily detected in current practice with ultrasound. In primary care centers, a positive test result may support the initiation of first-line treatment for endometriosis, such as oral contraceptives, or trigger a referral to a specialist. An estimated 10% of women presenting with dysmenorrhea have secondary dysmenorrhea, mostly due to endometriosis

[52] . In this population, the 1.2 kb and 3.7 kb deletions are highly effective in excluding endometriosis, with a negative predictive value (NPV) of 97%. In secondary care centers, a positive test result may guide the decision to initiate second-line treatment, such as gonadotropin-releasing hormone antagonists, or to proceed with laparoscopic surgery. Importantly, in the former center, the risk associated with false-positive results is less important, so a diagnostic cutoff value can be chosen to maximize the sensitivity of the test, while in the latter center, a different diagnostic cutoff value may be beneficial to maximize specificity and minimize the exposure of disease-free women to the risks associated with these interventions.

[0202] In addition to diagnostic accuracy, the ability of these two biomarkers to detect endometriosis did not correlate with patient age, sample age, or hormonal status, and only the 3.7 kb deletion showed a slight correlation with the phase of the patient's menstrual cycle at the time of sample collection. Further studies are needed to confirm whether this correlation with menstrual phase exists in a larger patient cohort or is an artifact of the number of samples used in this study. We demonstrated that both the 1.2 kb and 3.7 kb deletions accurately detect all subtypes and stages of the disease. In contrast to the current diagnostic standard, which includes visualization during surgery and possible resection for histological confirmation, mtDNA deletion-based assays require only a blood sample and can provide objective results before or in place of surgical intervention. Thus, if successfully translated into clinical use, mtDNA-based assays could reduce diagnostic delays

[16] and provide actionable results earlier in the course of the disease than previously possible.

[0203] From a practical standpoint, the use of blood-based biomarker assays has several advantages that effectively improve upon the current standard of care. Samples are easy and inexpensive to collect via venipuncture, and there is a low likelihood of co-occurring disease states associated with collection. Blood samples can be easily collected in an outpatient physician's office or clinic, without requiring space and equipment dedicated to surgical procedures. As a result of the high copy number of mtDNA, standard DNA extraction methods can be used without concentration techniques, and sufficient DNA can be recovered from standard blood samples, so a low failure rate of the test can be expected. The assay uses cost-effective PCR-based techniques that are widely used in clinical laboratories, the assay is quantitative, and the results are easily interpreted (i.e., the test result is either above or below a clear diagnostic cutoff, corresponding to either a positive or negative result). Finally, the absence or minimal correlation with menstrual phase ensures that sample collection requirements are simplified, and menstrual timing does not need to be considered when scheduling venipuncture.

[0204] A key element in successful disease management is understanding disease epidemiology. Due to relatively complex diagnostic procedures and symptoms that overlap with other gynecologic diseases, the epidemiology of endometriosis is poorly characterized and varies by patient population and geographic location [1, 2, 4, 6]. With the advent of molecular assays such as those described herein, additional data may become more readily available and help fill some of the gaps in our understanding of endometriosis epidemiology. Importantly, this study utilized widely available, standardized methods for specimen collection and processing, which will allow for more direct comparison of test results across different studies and patient populations [41-44, 53].

[0205] Importantly, the location of the mtDNA deletions may aid in elucidating the pathophysiological process of endometriosis. Both the 1.2kb and 3.7kb deletions affect all or part of genes encoding complexes I and V (ATP synthase) of the respiratory chain and several tRNAs. These deletions likely result in abnormal mitochondrial ATP synthase and complex I proteins, but the heteroplasmic nature of mtDNA likely allows for some degree of functional compensation within the population. Interestingly, the best two candidates out of the seven tested in this study are deletions that lie within regions that overlap with each other in the mitochondrial genome. Given that the 1.2kb deletion region (ATP6~ND3) resides within the larger 3.7kb deletion (ATP6~ND5), it is perhaps not surprising that the diagnostic accuracy of the two deletions is similar.

[0206] Based on the criteria presented here, the 1.2 kb and 3.7 kb mtDNA deletions are associated with endometriosis; however, further testing is required to understand what mechanistic role this mitochondrial genomic region plays in the development of endometriosis.

[0207] Limitations of this study include the use of patient-reported hormone and menstrual status, which may be less accurate than employing study-specific data measures. The data are encouraging but require replication and validation in larger independent datasets. This study is currently ongoing.

[0208] summary Below is a summary of the studies mentioned above: Endometriosis is a significant health burden affecting up to 10% of women worldwide. Currently, diagnosis is based on surgical visualization followed by histological confirmation.

[0209] Diagnosis is often complicated by the variety of clinical manifestations and symptoms that overlap with other gynecological diseases. As a result, definitive diagnosis is delayed for up to 10 years, resulting in high mortality and poor quality of life for affected individuals.

[0210] Thus, there is a clear need for reliable and rapid diagnostic aids that can yield actionable results early in the disease process.

[0211] Study samples were collected from women scheduled to undergo laparoscopy for pelvic pain (symptomatic) or tubal ligation (asymptomatic). Study participants were women aged 18 years or older (premenopausal) and confirmed to be non-pregnant.

[0212] Seven candidate mtDNA deletions were identified and evaluated to determine whether each was detectable in plasma, had sufficient copy number for reliable detection, had the predicted amplicon size, was specific, did not co-amplify pseudogenes in the cell nucleus, or generated nonspecific amplification products.

[0213] The six candidate deletions were further evaluated by QPCR and clinical specimens to determine whether each met the criteria for a sound diagnostic assay and to assess their accuracy in distinguishing endometriosis specimens from control specimens. Two deletions (1.2kb and 3.7kb deletions) were selected as promising biomarker candidates.

[0214] The 1.2kb and 3.7kb deletions accurately detected endometriosis, including all subtypes and stages, and detection did not correlate with patient or specimen age or hormonal therapy. The 3.7kb deletion correlated significantly with menstrual phase, but only in two phases.

[0215] Biomarkers derived from the mitochondrial genome containing the 1.2 kb and 3.7 kb deletions described here provide a promising and largely unexplored avenue in the search for diagnostic markers for endometriosis that can be effectively translated into clinical use.

[0216] Based on minimally invasive specimens, assays based on these markers could positively impact the diagnostic landscape for endometriosis by shortening diagnostic delays and providing actionable, objective and rapid test results.

[0217] conclusion Biomarkers derived from the mitochondrial genome, especially the 1.2kb and 3.7kb deletions described herein, provide a promising and largely unexplored avenue in the search for diagnostic markers for endometriosis that can be effectively transferred to clinical use. Based on minimally invasive specimens, assays based on these markers can be found to accurately diagnose endometriosis in blood samples from patients. Thus, this description provides a rapid, accurate and effective means for diagnosing endometriosis, thereby shortening the delay in obtaining a diagnosis and administering the necessary treatment protocol. This description allows a targeted diagnosis to be made for one or more of the mtDNA deletions (including either the large or small sublimon) and any fusion transcripts resulting therefrom. The same conclusion can be extended to any translation product resulting from the fusion transcript.

[0218] Example 3: Identification of the 8.7 kb mtDNA deletion for detection of endometriosis In this study, we identified an 8.7 kb deletion (deletion ID number 2767) using a combination of next-generation sequencing (NGS) and proprietary data mining software in a set of 10 cases and 10 controls obtained from Fidelis Research (Sofia, Bulgaria). The methods used for this identification are described in more detail in Example 4. We directly detected this biomarker in endometriosis tissue lesions using both qPCR and NGS. We selected the 8.7 kb deletion for evaluation based on its sequence composition, the presence of flanking repeat positions within the major arc of the mitochondrial genome where relatively more deletions have been reported

[47] , and our observations in endometrial tissues. Data from this study are shown in Table 14 and Figures 9-11.

[0219] [Table 14]

[0220] The 8.7 kb deletion removes all or part of the gene between NADH dehydrogenase subunits 2 and 5. An initial round of standard (quantitative) PCR and visualization after gel electrophoresis was used to prequalify each of the deletion targets and determine whether each candidate was (i) detectable; (ii) in sufficient copy number for reliable detection; (iii) had the expected amplicon size; and (iv) specific and did not co-amplify with nuclear pseudogenes or generate non-specific amplification products.

[0221] We successfully detected the deletion in circulating plasma and further evaluated by qPCR to determine whether the target was detectable in rho 0 cells using more sensitive qPCR. We also evaluated whether the assay had sufficient diagnostic accuracy and acceptable precision (defined as a maximum deviation of 1.5 Ct between at least two of the three replicates).

[0222] Further investigation of this deletion is described in Example 4.

[0223] Example 4: 8.7 kb mtDNA deletion to detect endometriosis in plasma of symptomatic women In this example, an 8.7 kb mtDNA deletion (FUS 5362:14049) was investigated as a potential biomarker for diagnosing endometriosis, including (i) an initial assessment of diagnostic accuracy, followed by ii) an assessment of disease specificity by comparing the frequency of the biomarker in plasma from women with: endometriosis controls and symptomatic controls, as well as endometrial, ovarian, and breast cancer.

[0224] method Diagnostic accuracy – Participants and sample collection This was a case-control study of prospectively collected residual plasma samples from non-pregnant women aged 18 years or older (premenopausal), scheduled to undergo laparoscopy for suspected endometriosis due to pelvic pain (symptomatic controls and endometriotic cases) or tubal ligation (asymptomatic controls). The study was conducted as part of the EndOx trial at the Oxford Endometriosis Medical Centre, John Radcliffe Hospital, University of Oxford, UK.

[0225] Collection, anonymization and processing of samples and data were as previously reported [26,41-44,57]. Study conduct, approval by the relevant authorities (Oxfordshire REC A, 09 / H0604 / 58) and consent procedures were also as previously reported [26,41-44;57].

[0226] Diagnostic accuracy – Participant population / cohort Collected samples were classified as either control or case samples: a) asymptomatic controls, which were specimens collected from participants undergoing planned tubal ligation without clinical suspicion of endometriosis and surgically confirmed to be free of endometriosis, and b) symptomatic controls, which were collected from participants with pain or other symptoms (except infertility) accompanied by clinical suspicion of endometriosis and no endometriotic lesions visualized laparoscopically by an experienced gynaecological surgeon.

[0227] The case group consisted of specimens in which the presence of endometriosis was diagnosed during laparoscopy and classified by the operating surgeon using the revised American Society for Reproductive Medicine (rASRM) stage (I: minimal; II: mild; III: moderate; IV: severe).

[45] Specimens were also classified by disease subtype: peritoneal, ovarian, and deeply infiltrating (DI) endometriosis.

[0228] Disease specificity - participants and sample collection Endometrial cases and controls based on diagnostic accuracy assessment were used to assess disease specificity and were compared with the remaining plasma samples obtained from OBIO (El Segundo, USA) and the Ontario Tumor Bank (Toronto, Canada).

[0229] Sample handling, processing, and mtDNA amplification Blood collection and processing Whole blood was collected into 10 ml K2EDTA Vacutainers® (BD Medical p / n BD366643) and centrifuged within 1 hour of collection at 2500×g for 10 minutes at 4° C. The plasma layer was removed, aliquoted, and stored at −80° C. until DNA extraction.

[0230] DNA extraction Total deoxyribonucleic acid (DNA) was extracted from plasma (200 μL) using a QIAamp™ 96 QIAcube™ HT extraction kit (Qiagen, Crawley, UK) automated by a QIAcube™ HT system (Qiagen, Crawley, UK), and extracted DNA was eluted with Buffer AE (200 μL).

[0231] qPCR for mtDNA deletions and standardization of qPCR with 18s rRNA For both real-time polymerase chain reaction (qPCR) procedures, we performed amplifications in 20 μL reactions using 96-well microplates (Bio-Rad, Hemel Hempstead, UK) containing non-standardized DNA template (5 μL), SYBR® Green master mix, and 250 nM of each primer for the 8.7 kb deletion and 18S ribosomal ribonucleic acid (rRNA) in each well. The primers used are shown in Table 15.

[0232] [Table 15]

[0233] For quantitative polymerase chain reaction (QPCR) with SYBR Green I fluorescence, a CFX96 Touch real-time PCR detection system (Bio-Rad, Hemel Hempstead, UK) was used.

[0234] Cycling conditions for the 8.7 kb deletion and 18S rRNA were 45 cycles of 95°C for 30 s, 66°C for 30 s, and 72°C for 30 s. After amplification, we performed melting curve analysis from 70°C to 90°C with readings every 0.5°C. Each plate of samples and controls was amplified in triplicate in triplicate.

[0235] quality control Quality control was performed as previously described

[56] . Briefly, quantification cycles (Cq) were calculated and the Cq of the deletion amplicon was normalized to the Cq of the multicopy nuclear target, the 18s rRNA gene amplicon. We amplified all samples in triplicate on separate plates. Two no-template control samples were processed together with each batch of DNA extraction and were confirmed as negative for amplification of both the deletion target and the 18S rRNA gene.

[0236] Rho 0 cell preparation Rho 0 cells were prepared as previously described [46; Creed 2019]. Briefly, cells derived from the human osteosarcoma cell line 143B (ATCC CRL 8303) were treated with ethidium bromide to deplete cytoplasmic mtDNA. Cells were grown to confluence in high glucose Dulbecco's modified Eagle's medium containing pyruvate, L-glutamine, uridine (50 μg / mL) and 5% fetal bovine serum.

[0237] statistical analysis No formal sample size calculation was performed, and the number of clinical specimens used was deemed sufficient to meet the study objectives. For qPCR, targets were amplified in triplicate from all specimens, and mean Cq values ​​were calculated. We determined standardized deletion values ​​(ΔCq) by quantifying deletion amplicons relative to the 18S rRNA reference amplicon. Statistical analysis was performed using Graphpad Prism™ 5.0 (Graphpad Software Inc, La Jolla, CA, USA) for ROC, descriptive statistics, correlation, and significance tests. We summarized clinical characteristics using counts and percentages for categorical data, and means, standard deviations (SD), and ranges for continuous variables. We summarized clinical characteristics using counts and percentages for categorical data, and means, standard deviations (SD), and ranges for continuous variables. Means of the two groups were compared using Student's t-test and Mann-Whitney U-test for parametric and nonparametric distributions, respectively. Correlation between two variables was assessed by Spearman correlation (r) or Mann Whitney U test or Kruskal-Wallis test. Receiver operating characteristic curves were constructed for the presence of endometriosis for all but the 6.5 kb deletion. The area under the ROC curve (AUC) as well as the sensitivity and specificity at the selected cutoffs were calculated with 95% confidence intervals (CI). For all tests, a p-value <0.05 was considered statistically significant.

[0238] result Study population and clinical samples Demographic and clinical characteristics for participants who provided samples are summarized in Table 16.

[0239] [Table 16]

[0240] Abbreviations: N = number of participants / sample; SD = standard deviation. (1) Means (SD) are shown; means and SD were calculated for participants who provided their age at the time of sample collection. (2) Participants' status within 3 months of sample collection. (3) Participants' menstrual status at the time of sample collection.

[0241] Overall, the mean (SD) ages of the control and case groups were statistically significantly different: 37.2 (6.9) years and 33.8 (6.8) years, p=0.0124. The majority of participants (121; 66.5%) had not received hormone therapy within 3 months prior to sample collection. Most participants who reported not menstruating were taking hormones (20 / 26; 76.9%).

[0242] Of the 182 samples collected, 32 were from the control group, 18 (9.49%) from symptomatic participants, and 14 (7.7%) from asymptomatic participants. The remaining 150 samples were from the case group, with 52 participants (28.6%) with peritoneal endometriosis, 48 ​​participants (26.4%) with ovarian endometriosis, 50 participants (27.5%) with DI endometriosis, and 91 (60.7%) classified as rASRM stage I / II disease and 58 (31.9%) as stage III / IV disease. 178 of the 182 samples (97.8%) showed valid assay results (2 peritoneal endometriosis samples and 1 ovarian endometriosis sample), and one symptomatic control sample was invalid for statistical analysis due to an out-of-range 18S rRNA Cq.

[0243] Preliminary evaluation of the 8.7 kb mtDNA deletion - standard PCR As described above in Example 3, we previously identified an 8.7 kb deletion in a set of 10 cases and 10 controls using a combination of next generation sequencing (NGS) and proprietary data mining software. As described above, we successfully detected the deletion in circulating plasma and performed further evaluation by qPCR to determine whether the target was detectable in rho 0 cells using the more sensitive qPCR.

[0244] Diagnostic accuracy of the 8.7 kb deletion Having successfully detected the 8.7 kb deletion in circulating plasma and endometriotic lesions, we investigated whether the 8.7 kb deletion could distinguish between symptomatic controls versus all endometriosis; between the three subtypes; and between the revised American Society for Reproductive Medicine (r-ASRM) classification stages in plasma from clinical samples from a larger population. We performed analyses using primarily samples from symptomatic controls and participants with confirmed disease to more accurately reflect the clinically relevant patient population (i.e., all those with endometriosis symptoms). We also measured the frequency of the deletion in asymptomatic control samples, and found no difference in the 8.7 kb deletion between symptomatic and asymptomatic control samples (p=0.681).

[0245] Symptomatic control versus total disease The 8.7 kb assay was used to provide good discrimination between symptomatic controls and all endometriosis specimens. The AUC (95% CI) of 0.8007 (0.7035-0.8979) was statistically significant (p<0.0001). We examined the ROC coordinates and selected a threshold to optimize sensitivity (a threshold of 6.650 differentiated between symptomatic controls and all subtypes / stages of endometriosis and showed acceptable sensitivity and specificity values ​​(Table 17)).

[0246] [Table 17]

[0247] Detection of disease by subtype It is important to be able to accurately detect all disease subtypes of endometriosis. In our study, the 8.7 kb deletion assay distinguished between samples from symptomatic controls and samples from patients with peritoneal endometriosis, ovarian endometriosis, and DI endometriosis (Figures 13A-13D). The mean (SD) ΔCt values ​​were 6.724 (1.192) for asymptomatic controls, 6.908 (1.26) for symptomatic controls, 4.086 (2.134) for peritoneal disease, 5.283 (1.801) for ovarian disease, and 5.617 (1.767) for DI endometriosis. Furthermore, the difference in the amount of the standardized 8.7 kb deletion between symptomatic controls was statistically significant for peritoneal endometriosis (p<0.0001), ovarian endometriosis (p=0.0002), and DI endometriosis (p=0.0023).

[0248] The diagnostic accuracy of the 8.7 kb deletion assay was also evaluated for each subtype (Figures 13A-13D). We correctly distinguished specimens from symptomatic controls and disease subtypes: AUC (95% CI) was 0.8882 (0.8043-0.9722; p<0.0001) for detection of peritoneal disease, 0.7766 (0.6572-0.8960; p=0.0008) for ovarian, and 0.7359 (0.6057-0.8661; p=0.0039) for DI endometriosis. Furthermore, a threshold of 6.65 showed acceptable sensitivity and specificity values ​​for distinguishing symptomatic controls vs. peritoneal endometriosis, ovarian disease, and DI disease (Table 17).

[0249] Detection of disease by stage of r-ASRM Endometriotic cases were classified into two stage groups: r-ASRM stage I / II and stage III / IV, and we examined whether both low-stage and high-stage disease could be accurately identified using the 8.7 kb deletion. The 8.7 kb deletion assay discriminated samples from symptomatic controls and samples from low-stage (stage I / II) and high-stage (stage III / IV) patients (Figures 14A-14C), with mean (SD) ΔCt values ​​of 6.908 (1.26) for symptomatic controls, 4.614 (2.063) for low-stage disease, and 5.565 (1.794) for high-stage disease.

[0250] Diagnostic accuracy assessed by receiver operating curves was highest for stages I / II: AUC 0.8361 (0.7426 to 0.9295; p < 0.0001) compared with stage III / IV: AUC 0.7465 (0.6232 to 0.8697; p = 0.0021). At a threshold of 6.65, sensitivity and specificity for all stages were acceptable (Table 17).

[0251] Correlation with patient age, specimen age, hormone therapy, and menstrual phase In an ideal assay, diagnostic accuracy would not be affected by factors such as patient and specimen age, hormonal therapy, and menstrual phase. We found no correlation between ΔCt values ​​and patient age (p=0.749) or between ΔCt values ​​and specimen age by age of collection (p=0.222) (Table 3). Similarly, no statistically significant differences in ΔCt values ​​were found when participants were stratified by hormonal status (p=0.838) or menstrual phase (p=0.233) (Table 18).

[0252] [Table 18]

[0253] The Mann-Whitney U-Test was used to determine the effect of hormonal status on endometriosis detection. The Kruskal Wallis was used to examine the effect of menstrual cycle on endometriosis detection.

[0254] Evaluation of disease specificity of the 8.7 kb deletion for endometriosis To further evaluate whether other female diseases show elevated levels of the 8.7 kb deletion, plasma samples from women subsequently diagnosed with endometrial cancer (n=12), ovarian cancer (n=72), and breast cancer (n=51) were obtained and compared to marker frequencies for plasma samples of three endometriosis subtypes (peritoneal, ovarian, and deep infiltrating endometriosis) and symptomatic controls (Figures 14A-14C). Significantly lower 8.7 kb deletions were detected in all three cancers (p<0.0001), measuring less than 64-fold detection in endometriotic cancers, less than 16-fold detection in ovarian cancers, and less than 8-fold detection in breast cancers compared to endometriosis. Results based on this evaluation are shown in Table 19.

[0255] [Table 19]

[0256] Data from Table 19 is shown in Figure 15. Figure 15 shows the normalized 8.7 kb deletion distribution for samples from endometrial cancer, ovarian cancer, breast cancer, symptomatic controls, and participants with peritoneal, ovarian or deep infiltrating endometriosis.

[0257] Consideration In this study, we demonstrated the utility of measuring 8.7 kb deletion biomarker levels in plasma samples as a potential assay for detecting endometriosis. Our assay met sound criteria for diagnostic testing, utilized a minimally invasive sample from blood, and accurately detected all subtypes and stages of disease, with the best track record seen in the peritoneal subtype and lower stages of endometriosis, both of which are frequently encountered in primary care settings. When translated into clinical use, this assay may shorten the time to diagnosis and allow medical intervention before surgery. Samples are easy and inexpensive to collect via venipuncture, and are unlikely to be associated with concurrent disease states.

[0258] With good diagnostic accuracy, especially for low-stage and peritoneal disease, the 8.7 kb deletion assay may augment current standard medical care, especially for low-stage and peritoneal disease, which cannot be reliably detected by imaging modalities, unlike ovarian and deep infiltrating endometriosis. The relative simplicity of the 8.7 kb deletion assay means that it may be feasible for use in both primary and secondary care facilities. Blood samples are routinely collected in primary care, without the need for dedicated surgical space or equipment. The high copy number of mtDNA means that standard DNA extraction methods can be used without concentration procedures. Furthermore, the sufficient amount of DNA recovered from a standard blood specimen does not indicate a high failure rate for the test. Real-time PCR-based techniques are used in clinical laboratories, which produce quantitative results that are easily interpreted.

[0259] In our study, we showed that there was no correlation between deletions and patient age, specimen age, hormonal status, or menstrual cycle phase. The lack of correlation with menstrual phase simplifies the requirement to collect samples without considering menstrual phase when scheduling sample collection.

[0260] The current complex diagnostic process, coupled with symptoms that overlap with other gynaecological conditions, means that the epidemiology of endometriosis is poorly characterized, which is a key factor in the successful management of any condition [1,2,4,6]. The emergence of molecular assays and novel biomarkers will provide further, more readily available data that will help improve understanding of endometriosis epidemiology. Importantly, our study used widely available standardized methods of specimen collection and processing, allowing for more direct comparison of test results across trials and patient populations [41-44,53].

[0261] conclusion The above-mentioned assay using the mitochondrial 8.7kb deletion biomarker is a blood sample-based, minimally invasive method for diagnosing endometriosis, which can be used in both primary and secondary care facilities.The relatively simple and patient-friendly approach provided by this assay shortens the time to diagnosis, thereby improving the management of the debilitating condition described herein, thereby improving the quality of life of patients.

[0262] Example 5: Identification of the 4.8 kb mtDNA deletion for detection of endometriosis A study similar to that described above was conducted to identify the frequency of the 4.8 kb mtDNA deletion (deletion ID 8590) and its correlation with endometriosis. For this analysis, the method of Example 4 was followed. The primers used in this study are shown in Table 20.

[0263] [Table 20]

[0264] Data demonstrating the utility of the 4.8 kb deletion in detecting endometriosis are provided in Table 21 and shown in Figures 12-14.

[0265] [Table 21]

[0266] Although the above description includes references to certain specific embodiments, various modifications of the embodiments will be apparent to those skilled in the art. Any examples provided herein are included for illustrative purposes only and are not intended to be limiting in any way. Any drawings provided herein are only for illustrating various aspects of the description and are not intended to be limiting or to scale in any way. The claims appended hereto should not be limited by the preferred embodiments set forth in the description above, but should be given the broadest interpretation consistent with the specification as a whole. The disclosures of all documents cited herein are incorporated herein by reference in their entirety.

[0267] [References] [1]Eskenazi B, Warner ML. Epidemiology of endometriosis. Obstet. Gynecol. Clin. North Am. 24(2), 235-258 (1997). [2]Kjerulff KH, Erickson BA, Langenberg PW. Chronic gynecological conditions reported by US women: findings from the National Health Interview Survey, 1984 to 1992. Am J Public Health. 86(2), 195-199 (1996). [3]Houston DE, Noller KL, Melton LJ, Selwyn BJ, Hardy RJ. Incidence of pelvic endometriosis in Rochester, Minnesota, 1970-1979. Am. J. Epidemiol. 125(6), 959-969 (1987). [4]Wheeler JM. Epidemiology of endometriosis-associated infertility. J Reprod Med. 34(1), 41-46 (1989). [5]Giudice LC, Kao LC. Endometriosis. Lancet. 364(9447), 1789-1799 (2004). [6]Vessey MP, Villard-Mackintosh L, Painter R. Epidemiology of endometriosis in women attending family planning clinics. BMJ. 306(6871), 182-184 (1993). [7] Zondervan KT, Becker CM, Koga K, Missmer SA, Taylor RN, Vigano P. Endometriosis. Nature Reviews Disease Primers. 4(1), 9 (2018). [8]Barlow DH, Kennedy S. Endometriosis: new genetic approaches and therapy. Annu. Rev. Med. 56, 345-356 (2005). [9]Giudice LC. Clinical practice. Endometriosis. N. Engl. J. Med. 362(25), 2389-2398 (2010).

[10] Fassbender A, Vodolazkaia A, Saunders P, et al. Biomarkers of endometriosis. Fertil. Steril. 99(4), 1135-1145 (2013). **Review of recent research in biomarkers for endometriosis

[11] SK, Kantawala KP, Menias CO. Beyond the boundaries-endometriosis: typical and atypical locations. Curr Probl Diagn Radiol. 40(6), 219-232 (2011).

[12] Berkley KJ, Rapkin AJ, Papka RE. The pains of endometriosis. Science. 308(5728), 1587-1589 (2005).

[13] Nnoaham KE, Hummelshoj L, Webster P, et al. Impact of endometriosis on quality of life and work productivity: a multicenter study across ten countries. Fertil. Steril. 96(2), 366-373.e8 (2011).

[14] Simoens S, Dunselman G, Dirksen C, et al. The burden of endometriosis: costs and quality of life of women with endometriosis and treated in referral centres. Hum. Reprod. 27(5), 1292-1299 (2012).

[15] Dunselman G a. J, Vermeulen N, Becker C, et al. ESHRE guideline: management of women with endometriosis. Hum. Reprod. 29(3), 400-412 (2014).

[16] Hadfield R, Mardon H, Barlow D, Kennedy S. Delay in the diagnosis of endometriosis: a survey of women from the USA and the UK. Hum. Reprod. 11(4), 878-880 (1996).

[17] Ahn SH, Singh V, Tayade C. Biomarkers in endometriosis: challenges and opportunities. Fertil. Steril. 107(3), 523-532 (2017). **Review of recent research in biomarkers for endometriosis

[18] Rifai N, Gillette MA, Carr SA. Protein biomarker discovery and validation: the long and uncertain path to clinical utility. Nat. Biotechnol. 24(8), 971-983 (2006).

[19] Hu C, van der Heijden R, Wang M, van der Greef J, Hankemeier T, Xu G. Analytical strategies in lipidomics and applications in disease biomarker discovery. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 877(26), 2836-2846 (2009).

[20] Liotta LA, Petricoin EF. Serum peptidome for cancer detection: spinning biologic trash into diagnostic gold. J. Clin. Invest. 116(1), 26-30 (2006).

[21] Petricoin EF, Ardekani AM, Hitt BA, et al. Use of proteomic patterns in serum to identify ovarian cancer. Lancet. 359(9306), 572-577 (2002).

[22] Petricoin EF, Belluco C, Araujo RP, Liotta LA. The blood peptidome: a higher dimension of information content for cancer biomarker discovery. Nat. Rev. Cancer. 6(12), 961-967 (2006).

[23] Zhang Z, Bast RC, Yu Y, et al. Three biomarkers identified from serum proteomic analysis for the detection of early stage ovarian cancer. Cancer Res. 64(16), 5882-5890 (2004).

[24] Anderson NL, Anderson NG. The human plasma proteome: history, character, and diagnostic prospects. Mol. Cell Proteomics. 1(11), 845-867 (2002).

[25] May KE, Conduit-Hulbert SA, Villar J, Kirtley S, Kennedy SH, Becker CM. Peripheral biomarkers of endometriosis: a systematic review. Hum. Reprod. Update. 16(6), 651-674 (2010).

[26] Rahmioglu N, Fassbender A, Vitonis AF, et al. Harmonization of Clinical and Laboratory Data to Improve Biomarker Discovery in Endometriosis: WERF EPHect. In: Biomarkers for Endometriosis. Springer, Cham, 199-226. **Summary of standardized methods in endometriosis research

[27] Taylor RW, Turnbull DM. Mitochondrial DNA mutations in human disease. Nat. Rev. Genet. 6(5), 389-402 (2005).

[28] Polyak K, Li Y, Zhu H, et al. Somatic mutations of the mitochondrial genome in human colorectal tumors. Nat. Genet. 20(3), 291-293 (1998).

[29] Petros JA, Baumann AK, Ruiz-Pesini E, et al. mtDNA mutations increase tumorigenicity in prostate cancer. Proc. Natl. Acad. Sci. USA 102(3), 719-724 (2005).

[30] Creed J, Klotz L, Harbottle A, et al. A single mitochondrial DNA deletion accurately detects significant prostate cancer in men in the PSA “grey zone.” World J Urol. 36(3), 341-348 (2018).

[31] Parrella P, Xiao Y, Fliss M, et al. Detection of mitochondrial DNA mutations in primary breast cancer and fine-needle aspirates. Cancer Res. 61(20), 7623-7626 (2001).

[32] Liang BC. Evidence for association of mitochondrial DNA sequence amplification and nuclear localization in human low-grade gliomas. Mutat. Res. 354(1), 27-33 (1996).

[33] Guo X-G, Guo Q-N. Mutations in the mitochondrial DNA D-Loop region occur frequently in human osteosarcoma. Cancer Lett. 239(1), 151-155 (2006).

[34] Alonso A, Martin P, Albarran C, et al. Detection of somatic mutations in the mitochondrial DNA control region of colorectal and gastric tumors by heteroduplex and single-strand conformation analysis. Electrophoresis. 18(5), 682-685 (1997).

[35] Liu VW, Shi HH, Cheung AN, et al. High incidence of somatic mitochondrial DNA mutations in human ovarian carcinomas. Cancer Res. 61(16), 5998-6001 (2001).

[36] Dai JG, Xiao YB, Min JX, Zhang GQ, Yao K, Zhou RJ. Mitochondrial DNA 4977 BP deletion mutations in lung carcinoma. Indian J Cancer. 43(1), 20-25 (2006).

[37] Liu VWS, Yang HJ, Wang Y, et al. High frequency of mitochondrial genome instability in human endometrial carcinomas. Br. J. Cancer. 89(4), 697-701 (2003).

[38] Wang Y, Xue WC, Liu VWS, Ngan HYS. Detection of mosaic pattern of mitochondrial DNA alterations in different populations of cells from the same endometrial tumor. Mitochondrion. 7(1-2), 171-175 (2007).

[39] Semczuk A, Lorenc A, Putowski L, et al. Clinicoprognostical features of endometrial cancer patients with somatic mtDNA mutations. Oncol. Rep. 16(5), 1041-1045 (2006).

[40] Pejovic T, Ladner D, Intengan M, et al. Somatic D-loop mitochondrial DNA mutations are frequent in uterine serous carcinoma. Eur. J. Cancer. 40(16), 2519-2524 (2004).

[41] Rahmioglu N, Fassbender A, Vitonis AF, et al. World Endometriosis Research Foundation Endometriosis Phenome and Biobanking Harmonization Project: III. Fluid biospecimen collection, processing, and storage in endometriosis research. Fertil. Steril. 102(5), 1233-1243 (2014). *Harmonized methods for endometriosis research

[42] Fassbender A, Rahmioglu N, Vitonis AF, et al. World Endometriosis Research Foundation Endometriosis Phenome and Biobanking Harmonization Project: IV. Tissue collection, processing, and storage in endometriosis research. Fertil. Steril. 102(5), 1244-1253 (2014). *Harmonized methods for endometriosis research

[43] Becker CM, Laufer MR, Stratton P, et al. World Endometriosis Research Foundation Endometriosis Phenome and Biobanking Harmonization Project: I. Surgical phenotype data collection in endometriosis research. Fertil. Steril. 102(5), 1213-1222 (2014). *Harmonized methods for endometriosis research

[44] Vitonis AF, Vincent K, Rahmioglu N, et al. World Endometriosis Research Foundation Endometriosis Phenome and Biobanking Harmonization Project: II. Clinical and covariate phenotype data collection in endometriosis research. Fertil. Steril. 102(5), 1223-1232 (2014). *Harmonized methods for endometriosis research

[45] American Society for Reproductive Medicine. Revised American Society for Reproductive Medicine classification of endometriosis: 1996. Fertility and Sterility. 67(5), 817-821.

[46] King MP, Attardi G. Human cells lacking mtDNA: repopulation with exogenous mitochondria by complementation. Science. 246(4929), 500-503 (1989).

[47] Brandon MC, Lott MT, Nguyen KC, et al. MITOMAP: a human mitochondrial genome database--2004 update. Nucleic Acids Res. 33(Database issue), D611-613 (2005).

[48] Stratton P, Winkel C, Premkumar A, et al. Diagnostic accuracy of laparoscopy, magnetic resonance imaging, and histopathologic examination for the detection of endometriosis. Fertil. Steril. 79(5), 1078-1085 (2003).

[49] Mettler L, Schollmeyer T, Lehmann-Willenbrock E, et al. Accuracy of laparoscopic diagnosis of endometriosis. JSLS. 7(1), 15-18 (2003). *Evaluation of the current gold standard for diagnosing endometriosis

[50] Falcone T, Mascha E. The elusive diagnostic test for endometriosis. Fertil. Steril. 80(4), 886-888 (2003).

[51] Wykes CB, Clark TJ, Khan KS. Accuracy of laparoscopy in the diagnosis of endometriosis: a systematic review. BJOG. 111(11), 1204-1212 (2004).

[52] Harel Z. Dysmenorrhea in adolescents and young adults: etiology and management. J Pediatr Adolesc Gynecol. 19(6), 363-371 (2006).

[53] Casper RF. Introduction: new tools for enhancing collaborative endometriosis research. Fertil. Steril. 102(5), 1211-1212 (2014).

[54] Salahi, E., et al.; Mitochondrial DNA 4977-bp deletion in endometriosis. Genes & Genomics, 35(5), 563-567 (2013).

[55] Taylor HS. Diagnosing endometriosis: Is laparoscopy the gold standard? Supplement to OBG Management. S1-S2 (2019).

[56] Ballard K, Lowton K, Wright J. What's the delay? A qualitative study of women's experiences of reaching a diagnosis of endometriosis. Fertil. Steril 86(5), 1296-301 (2006).

[57] Creed J, Maggrah A, Reguly B, Harbottle A. Mitochondrial DNA deletions accurately detect endometriosis in symptomatic females of child-bearing age. Biomark. Med. 13(4), 291-306 (2019).

[58] The Practice Committee of the American Society for Reproductive Medicine. Endometriosis and infertility. Fertil. Steril. 81, 1441-1446 (2004).

[59] Nicholls, T.J., et al., Linear mtDNA fragments and unusual mtDNA rearrangements associated with pathological deficiency of MGME1 exonuclease. Hum Mol Genet., 23(23):6147-62 (2014).

[60] Zhang C. et al., Multiple mitochondrial DNA deletions in an elderly human individual. FEBS, 297, 1, 2, 34-38 (1992).

[61] Wei, Y.H., Mitochondrial DNA alterations as ageing-associated molecular events. Mutat. Res., 275(3-6), 145-55 (1992).

[62] Tanaka, M. et al., Direct sequencing of deleted mitochondrial DNA in myopathic patients. Biochem Biophys Res Commun., 164(1):156-63, (1989).

[63] Kapsa, R. et al., A novel mtDNA deletion in an infant with Pearson syndrome. J Inherit Metab Dis., 17(5):521-6, (1974).

[64] Hseih, R.H., et al., Multiple rearrangements of mitochondrial DNA in unfertilized human oocytes. Fertil Steril., 77(5):1012-7, (2002).

[65] Hjelm, BE et al., http: / / mitobreak.portugene.com / cgi-bin / Mitobreak_individual_page.cgi?Name=5371:14058&Breaktype=Del&Species=Homsap&Published=Pub [Brief description of the drawings]

[0268] [Figure 1] FIG. 1 shows mitochondrial coding genes. [Figure 2A] 1 shows detection of the fusion transcript in endometrial tissue. [Figure 2B] 1 shows detection of the fusion transcript in endometrial tissue. [Figure 2C] 1 shows detection of the fusion transcript in endometrial tissue. [Figure 2D] 1 shows detection of the fusion transcript in endometrial tissue. [Figure 2E] 1 shows detection of the fusion transcript in endometrial tissue. [Figure 2F] 1 shows detection of the fusion transcript in endometrial tissue. [Figure 2G] 1 shows detection of the fusion transcript in endometrial tissue. [Figure 2H] 1 shows detection of the fusion transcript in endometrial tissue. [Figure 2I] 1 shows detection of the fusion transcript in endometrial tissue. [Figure 2J] 1 shows detection of the fusion transcript in endometrial tissue. [Diagram 3] The mtDNA fusion transcript map is shown. [Figure 4A] 1 shows the diagnostic accuracy of the 1.2 kb and 3.7 kb deletions in Example 2. [Figure 4B] 1 shows the diagnostic accuracy of the 1.2 kb and 3.7 kb deletions in Example 2. [Figure 5A] FIG. 1 shows the diagnostic accuracy of the 1.2 kb deletion of Example 2 in discriminating between symptomatic control samples and samples of different endometrial disease subtypes. [Figure 5B] FIG. 1 shows the diagnostic accuracy of the 1.2 kb deletion of Example 2 in discriminating between symptomatic control samples and samples of different endometrial disease subtypes. [Figure 5C] FIG. 1 shows the diagnostic accuracy of the 1.2 kb deletion of Example 2 in discriminating between symptomatic control samples and samples of different endometrial disease subtypes. [Figure 5D] FIG. 1 shows the diagnostic accuracy of the 1.2 kb deletion of Example 2 in discriminating between symptomatic control samples and samples of different endometrial disease subtypes. [Figure 6A] 1 shows the diagnostic accuracy of the 3.7 kb deletion of Example 2 in distinguishing between symptomatic control samples and samples of endometrial disease subtypes. [Figure 6B] 1 shows the diagnostic accuracy of the 3.7 kb deletion of Example 2 in distinguishing between symptomatic control samples and samples of endometrial disease subtypes. [Figure 6C] 1 shows the diagnostic accuracy of the 3.7 kb deletion of Example 2 in distinguishing between symptomatic control samples and samples of endometrial disease subtypes. [Figure 6D] 1 shows the diagnostic accuracy of the 3.7 kb deletion of Example 2 in distinguishing between symptomatic control samples and samples of endometrial disease subtypes. [Figure 7A] 1 shows the diagnostic accuracy of the 1.2 kb deletion of Example 2 in distinguishing symptomatic control samples from samples from patients with known disease stages. [Figure 7B] 1 shows the diagnostic accuracy of the 1.2 kb deletion of Example 2 in distinguishing symptomatic control samples from samples from patients with known disease stages. [Figure 7C] 1 shows the diagnostic accuracy of the 1.2 kb deletion of Example 2 in distinguishing symptomatic control samples from samples from patients with known disease stages. [Figure 8A]1 shows the diagnostic accuracy of the 3.7 kb deletion of Example 2 in distinguishing between symptomatic control samples and samples from patients with known disease stage. [Figure 8B] 1 shows the diagnostic accuracy of the 3.7 kb deletion of Example 2 in distinguishing between symptomatic control samples and samples from patients with known disease stage. [Figure 8C] 1 shows the diagnostic accuracy of the 3.7 kb deletion of Example 2 in distinguishing between symptomatic control samples and samples from patients with known disease stage. [Figure 9] FIG. 11 is a scatter plot showing the difference in 8.7 kb deletion scores between endometriosis positive samples, symptomatic control samples and normal healthy control samples. [Figure 10] Box plot showing the difference in 8.7 kb deletion scores between endometriosis positive samples, symptomatic control samples and normal healthy control samples. [Figure 11] 4 shows the ROC curve for the 8.7 kb deletion comparing endometriosis positive patients vs. healthy / normal controls. [Figure 12] Diagnostic accuracy of the 8.7 kb deletion - syndromic vs. all endometrial disease - is shown. [Figure 13A] Diagnostic accuracy of the 8.7 kb deletion - control vs. disease - by subtype is shown. [Figure 13B] The area under the ROC curve was calculated to indicate diagnostic accuracy. [Figure 13C] The area under the ROC curve was calculated to indicate diagnostic accuracy. [Figure 13D] The area under the ROC curve was calculated to indicate diagnostic accuracy. [Figure 14A] Diagnostic accuracy of the 8.7 kb deletion - control vs. disease by stage - is shown. [Figure 14B] Diagnostic accuracy of the 8.7 kb deletion - control vs. disease by stage - is shown. [Figure 14C] Diagnostic accuracy of the 8.7 kb deletion - control vs. disease by stage - is shown. [Figure 15] This shows the disease specificity of the 8.7 kb deletion to endometriosis. [Figure 16] FIG. 11 is a scatter plot showing the difference in scores of the 4.8 kb deletion between endometriosis positive samples, symptomatic control samples and normal healthy control samples. [Figure 17] Box plot showing the difference in 4.8 kb deletion scores between endometriosis positive samples, symptomatic control samples and normal healthy control samples. [Figure 18] The ROC for the 4.8 kb deletion is shown, comparing data from endometriosis positive patients and symptomatic controls. [Figure 19] The ROC for the 4.8 kb deletion comparing data from endometriosis positive patients vs. healthy / normal controls is shown. [Figure 20] The occurrence of deletions according to the present specification is shown.

Claims

1. 1. A method for identifying an abnormal mitochondrial DNA (mtDNA) molecule having a nucleotide sequence having a deletion in a biological sample from a mammalian subject, comprising: Once recircularized, the mtDNA contains a junction point consisting of a first and a second nucleotide, With respect to SEQ ID NO:1: said deletion comprising nucleotides 5377 to 14048; the first nucleotide is between nucleotides 5362 and 5377, and the second nucleotide is between nucleotides 14048 and 14063; The method is for detecting and / or monitoring endometriosis in a mammalian subject, quantitating the amount of the abnormal mtDNA in the biological sample and comparing the quantified amount to a reference value; If the quantified amount is higher than the reference value, endometriosis is detected. method.

2. 2. The method of claim 1, wherein the first nucleotide is at position 5362 and the second nucleotide is at position 14049.

3. 3. The method of claim 1 or 2, wherein the abnormal mtDNA comprises the nucleotide sequence set forth in SEQ ID NO:

75.

4. The method of any one of claims 1 to 3, wherein said identifying comprises contacting said biological sample with: a) a nucleic acid probe having a nucleotide sequence complementary to a portion of the nucleotide sequence of the aberrant mtDNA that includes the junction point; b) a pair of nucleic acid primers, one of the primers having a nucleotide sequence complementary to a portion of the nucleotide sequence of the aberrant mtDNA that includes the junction point; or c) a pair of nucleic acid primers, each of said primers having a nucleotide sequence complementary to a nucleotide sequence of said aberrant mtDNA adjacent to said junction point.

5. 5. The method of claim 4, wherein said one of said primers of said primer pair has the nucleotide sequence set forth in SEQ ID NO:

83.

6. The method of claim 5 , wherein the other primer of the primer pair has the nucleotide sequence set forth in SEQ ID NO:

82.

7. A method for identifying in a biological sample from a mammalian subject a fusion transcript having a nucleotide sequence encoded by: a) at least a portion of an abnormal mitochondrial DNA (mtDNA) molecule having a deletion that creates a junction point after the mtDNA is recircularized, wherein the junction point is between nucleotides 5362:14049 of SEQ ID NO:1, and the portion includes the junction point; or b) at least a portion of an abnormal mitochondrial DNA (mtDNA) molecule as defined in any one of claims 1 to 3, wherein said portion includes said junction point; The method is for detecting and / or monitoring endometriosis in a mammalian subject, quantitating the amount of the fusion transcript in the biological sample and comparing the quantified amount to a reference value; If the quantified amount is higher than the reference value, endometriosis is detected.

8. The method of claim 7, comprising: a) contacting the biological sample with a nucleic acid probe having a nucleic acid sequence complementary to the nucleotide sequence of the fusion transcript having the transcribed junction point; or b) identifying the translation product of said fusion transcript.

9. the fusion transcript has the nucleotide sequence set forth in SEQ ID NO:77; or The method of claim 7 or 8, wherein the translation product has the amino acid sequence set forth in SEQ ID NO:

79.

10. The method of any one of claims 1 to 9, wherein the biological sample is blood, tissue, or menstrual fluid obtained from the subject.

11. 1. A method for identifying deleted mitochondrial DNA (mtDNA) molecules in a biological sample from a mammalian subject, comprising: the deletion comprises nucleotides 5362 to 14049 of SEQ ID NO:1; The method is for detecting and / or monitoring endometriosis in a mammalian subject, quantitating the amount of the deleted mtDNA in the biological sample and comparing the quantified amount to a reference value; If the quantified amount is higher than the reference value, endometriosis is detected.

12. The method of claim 11 , wherein the biological sample is blood, tissue, or menstrual fluid obtained from the subject.

13. A kit for carrying out the method according to any one of claims 1 to 12, comprising: The kit comprises at least one of the following: a) a pair of nucleic acid primers, one of the primers having a nucleotide sequence complementary to a portion of the nucleotide sequence of the aberrant mtDNA that includes the junction point; or b) a pair of nucleic acid primers, each of said primers having a nucleotide sequence complementary to a nucleotide sequence of said aberrant mtDNA adjacent to said junction point.

14. The kit of claim 13, comprising at least one of the following: a) primers and / or probes complementary to one or more fusion transcripts of said aberrant mtDNA molecule; or b) a binding agent adapted to bind to a protein encoded by said aberrant mtDNA molecule.

15. The kit of claim 14, wherein the binding agent is an antibody or an antigen-binding fragment.

Images