Mitochondrial DNA deletions associated with endometriosis

Abstract

To provide a method for detecting, diagnosing, or monitoring endometriosis in a sample.SOLUTION: The present invention provides a method for identifying aberrant mitochondrial DNA (mtDNA) molecules having deletions in a biological sample derived from a mammalian subject, wherein the deletions comprise sequences between specific nucleotides of a specific mtDNA nucleotide sequence, and wherein, when re-circularized, the mtDNA comprises a junction site.SELECTED DRAWING: Figure 1

Description

Detailed Description of the Invention

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

[0002] [Description of the Sequence Listing] The sequence listing related to this application is submitted herewith in ASCII format and is incorporated herein by reference. The title of the text file containing the sequence listing is "Sequence_listing.txt", created on December 17, 2019, and has a size of approximately 119 kilobytes.

[0003] [Technical Field to Which the Invention Belongs] This description generally relates to novel biomarkers and methods for detecting / diagnosing and / or observing endometriosis. This description also relates to unique specimens and / or reagents useful in the methods of this subject matter.

[0004] [Background] Endometriosis is a burdensome disease that occurs in up to 5 - 10% of women of reproductive age and is a common cause of infertility [1 - 7,58]. This disease is characterized by the presence of endometrial tissue (epithelial cells and stroma) that grows outside the uterus. Such ectopic endometrial tissue is found on the pelvic peritoneum and fallopian tubes, ovaries, intestines and bladder, and rarely in more distal body sites [8 - 11]. Women with endometriosis are often troubled by debilitating symptoms such as non - menstrual pelvic pain, painful menstrual cramps, pain during intercourse, fatigue, and infertility at high frequencies

[12] , which can lead to a significant decline in QOL

[13] . Considering its high prevalence and significant morbidity, endometriosis causes very substantial economic losses worldwide, estimated at tens of billions of euros annually

[14] .[[]END]]

[0005] Unfortunately, the diagnosis of endometriosis is often a long process, and as a result, treatment is often delayed. The current "gold standard" for diagnosing endometriosis is laparoscopy followed by histopathological confirmation of tissue specimens [5,15]. Making a timely diagnosis is further complicated by reporting delays

[16] and misinterpretation of symptoms

[17] , and can be further delayed if patients are reluctant to undergo expensive and invasive laparoscopic procedures. In fact, delays in the diagnosis of endometriosis can exceed 10 years

[16] . These delays can cause the majority of women to develop moderate to severe symptoms before a definitive diagnosis is made, resulting in increased morbidity, treatment costs, and a decline in QOL

[14] . Therefore, there is a need for a reliable, non-invasive test that can facilitate the early detection of endometriosis and provide actionable real-time results. However, non-invasive methods for detecting endometriosis are not currently available.

[0006] Molecular biomarkers are widely used as tools for measuring, detecting, and predicting human diseases [18-24]. However, it has been proven difficult to search for endometriosis-specific biomarkers

[25] . Some of the major issues include non-standardized specimen collection, analysis methods, and data interpretation, as well as a lack of biomarker specificity

[17] . In recent years, efforts have been made to harmonize the methods of collecting and preserving biological specimens and the reporting of endometriosis data, including the World Endometriosis Research Foundation (WERF) EPHect protocol

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

[0007] The mitochondrial genome represents a biomarker repository that has not been extensively explored. As shown in Figure 1, the mitochondrial genome encodes the complement of 24 genes, including two rRNAs and 22 tRNAs that ensure the accurate translation of the remaining 13 genes essential for electron transport. The targeted 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 mutation and deletion events can be reliably amplified from heteroplasmic mitochondrial populations. Indeed, mtDNA mutations have been well documented as biomarkers for several cancers across multiple body sites, including bone, brain, breast, lung, colorectal, stomach, ovary, prostate, and endometrial tissues [28 - 37]. While the nuclear genome has over three billion base pairs, the mitochondrial (mt) genome is relatively small, having 16,569 nucleic acid base pairs. Furthermore, considering the clonal expansion of mitochondria within the fertilized egg, all mtDNA genomes in a given individual are typically identical. The mt genome is also unique in that it is a circular, intron-free DNA molecule with repeat motifs scattered adjacent to sequences of a specific length. The sequences between these repeats tend to be deleted under poorly understood circumstances. Moreover, such deletions often include at least a portion of one or both of the flanking repeat sequences. As described further below, once the sequence that constitutes the deletion is removed, the remaining "parental" mtDNA re-circularizes to form a "major sub-monomer (sublimon)". Similarly, the deleted sequence may also re-circularize to form a "minor sub-monomer". Considering the number of repeats in the mt genome, there are many potential deletions. One of the best-known examples among these deletions is the 4977bp "common deletion" associated with various pathologies. Although the common deletion has also been investigated as a marker for endometriosis

[54] , it was not suggested that such deletions would be effective markers for this disease due to its lack of specificity.Certain mitochondrial DNA deletions have previously been associated with several specific conditions and age-related disorders (see

[59] -

[64] ). An 8,686 bp deletion between nucleotides 5371 and 14058 of the mtDNA genome has also been reported (

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

[0008] In some cases, mtDNA deletions and other large-scale mtDNA rearrangements can give rise to transcriptionally active mutant mtDNA sequences, resulting in mitochondrial fusion transcripts. Examples of the association between mitochondrial fusion transcripts and disease stage 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 the study of endometrial cancer, MtDNA modifications in the endometrium have been detected [37 - 40]. However, in these studies, no consensus region within the mtDNA genome or specific mtDNA modifications correlated with endometrial disease were revealed. As a result, these studies did not suggest that mtDNA modifications could be used as biomarkers for the detection of endometriosis. Furthermore, previous studies are thought not to have led to an association between mitochondrial fusion transcripts and endometrial disease or stage.

[0010] Therefore, there is a need for accurate and / or more effective means of detecting endometrial disease and / or condition that address at least one defect in known methods.

[0011] [Summary of the Explanation] In one aspect, the present description provides a method, reagent, and / or kit for detecting, diagnosing, and / or observing 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 being associated with endometriosis. The methods of the present invention can be carried out using biological samples obtained from a subject being screened. Such samples can include tissues (e.g., biopsy tissues), menstrual fluid, circulating blood, or blood derivatives such as serum or plasma. The methods currently described can be carried out on samples obtained non-invasively from a subject and serve as an effective means of determining whether a subject has or is suspected of having endometriosis and whether further invasive diagnostic investigations are necessary.

[0012] In one aspect, a method is provided for detecting, diagnosing, and / or observing endometriosis in a mammalian subject, 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 recombined or circularized mtDNA nucleotide sequence, the junction point being at nucleotide pair 8469:13447, 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 of the mtDNA nucleotide sequence of SEQ ID NO: 1.

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

[0014] In one aspect, the method includes the step of identifying a chimeric transcript of an abnormal mtDNA molecule.

[0015] In another aspect, the method includes the step of identifying a chimeric protein encoded by an abnormal mtDNA molecule.

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

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

[0018] In another aspect, a method for detecting fusion transcripts and fusion proteins resulting from abnormal mtDNA molecules or mtDNA deletions is provided.

[0019] [Brief Description of the Drawings] The features of certain embodiments will become more apparent in the following detailed description with reference to the accompanying drawings.

[0020] FIG. 1 is a diagram showing the coding genes of mitochondria.

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

[0022] Figure 3 shows a map of the mtDNA fusion transcript showing the mtDNA genome of SEQ ID NO: 1, the gene positions, and the positions of the 10 portions of the deleted mtDNA (i.e., “probes” or “targets”) described herein. The positions 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 endometrial disease states. The 1.2 kb and 3.7 kb deletions were evaluated for their ability to distinguish between symptomatic patient specimens and specimens from patients with confirmed endometriosis (all subtypes / stages combined). Receiver operating characteristic curves were generated and the area under the curve was calculated. Abbreviations: CI = confidence interval; ROC = receiver operating characteristic; Std = standard; vs = versus.

[0024] Figures 5A - 5D show the diagnostic accuracy of the 1.2 kb deletion of Example 2 in distinguishing between symptomatic control samples and samples of different endometrial disease subtypes. The 1.2 kb deletion was evaluated for its ability to distinguish between specimens from symptomatic patients and specimens from patients stratified by endometriosis subtypes (peritoneal, ovarian, deep infiltration). Figure 5A shows the distribution of the normalized 1.2 kb deletion for specimens from symptomatic controls and patients with endometriosis infiltrating the peritoneum, ovary, or deeply. The boundaries of the box are the 25 th and 75 th percentiles, the central line is 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 operating characteristic curves were generated for the 1.2 kb deletion 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 per group; ROC = receiver operating characteristic; Std = standard; vs = versus.

[0025] Figures 6A-6D show the diagnostic accuracy of the 3.7 kb deletion of Example 2 in distinguishing symptomatic control samples from samples of endometrial disease subtypes. The 3.7 kb deletion was evaluated for its ability to distinguish between specimens from symptomatic patients and specimens from patients stratified by subtypes of endometriosis (peritoneal, ovarian, deep infiltrating). Figure 6A shows the distribution of the standardized 3.7 kb deletion for symptomatic controls and sample specimens from patients with peritoneal, ovarian, or deep infiltrating endometriosis. The boundaries of the box represent the 25th and 75th percentiles, the central 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 6B-6D, receiver operator characteristic curves were generated for the 3.7 kb deletion, the area under the curve was calculated, and diagnostic accuracy was shown. Abbreviations: CI = confidence interval; Dev = deviation; DIE = deep infiltrating endometriosis; N = number of specimens per group; ROC = receiver operator characteristic; Std = standard; vs = versus.

[0026] Figures 7A-7C show 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. The 1.2 kb deletion was evaluated for its ability to distinguish between specimens from symptomatic patients and specimens from patients stratified by stage of endometriosis (low or high). Figure 7A shows the distribution of the standardized 1.2 kb deletion for symptomatic controls and specimens from patients with low (I / II) or high (III / IV) stages of endometriosis. The boundaries of the box represent the 25 th th percentile and 75 th th percentile, the central 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 specimens per group; ROC = receiver operator characteristic; Std = standard; vs = versus.

[0027] Figures 8A - 8C show the diagnostic accuracy of the 3.7 kb deletion of Example 2 in distinguishing symptomatic control samples from samples from patients with known disease stages. The 3.7 kb deletion was evaluated for its ability to distinguish between specimens from symptomatic patients and specimens from patients stratified by the stage (low or high) of endometriosis. Figure 8A shows the distribution of the normalized 3.7 kb deletion for specimens from symptomatic controls and patients with low (I / II) or high (III / IV) stages of endometriosis. The boundaries of the box represent the 25 th percentile and 75 th percentile, the central 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 8B and 8C, receiver operating characteristic (ROC) curves were created 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 specimens per group; ROC = receiver operating characteristic; Std = standard; vs = versus.

[0028] Figure 9 is a scatter plot showing the differences in 8.7 kb deletion scores among endometriosis - positive samples, symptomatic control samples, and normal healthy control samples.

[0029] Figure 10 is a box - and - whisker plot showing the differences in 8.7 kb deletion scores among endometriosis - positive samples, symptomatic control samples, and normal healthy control samples.

[0030] Figure 11 shows the ROC curve of the 8.7 kb deletion comparing endometriosis - positive patients vs healthy / normal controls.

[0031] Figure 12 shows the diagnostic accuracy of the 8.7 kb deletion for symptomatic vs. all endometrial diseases. The 8.7 kb deletion was evaluated for its ability to distinguish between samples from symptomatic patients and samples from patients with confirmed endometriosis (pooled across all subtypes / stages) by calculating the area under the ROC curve. Abbreviations: CI = confidence interval; ROC = receiver operating 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 between samples from symptomatic participants and samples from participants stratified by endometriosis subtypes (peritoneal, ovarian, deep infiltrating). Figure 13A shows the distribution of the standardized 8.7 kb deletion for specimens from asymptomatic and symptomatic controls, peritoneal, ovarian, or deep infiltrating endometriosis participants. The boundaries of the box represent the 25th and 75th 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 also summarized for each group. Figures 13B - 13D show the area under the ROC curve calculated to demonstrate diagnostic accuracy. Abbreviations: As Con = asymptomatic control; CI = confidence interval; Dev = deviation; DIE = deep infiltrating endometriosis; N = number of specimens per group; ROC = receiver operating characteristic; Sym Con = symptomatic control; Std = standard; vs = versus.

[0033] Figures 14A - 14C further show the diagnostic accuracy of the 8.7 kb deletion - control versus disease by stage. These figures show whether the 8.7 kb deletion assay can distinguish between samples from symptomatic participants and samples from participants stratified by endometriosis stages I / II and III / IV. Figure 14A shows the distribution of the standardized 8.7 kb deletion for specimens from symptomatic controls, participants with low (I / II) or high (III / IV) stages of endometriosis. The boundaries of the box represent the 25th and 75th 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. Figures 14B and 14C show the area under the ROC curve calculated to show diagnostic accuracy. Abbreviations: CI = confidence interval; Dev = deviation; N = number of specimens per group; ROC = receiver operator characteristic; Sym Con = symptomatic control; Std = standard; vs = versus.

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

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

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

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

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

[0039] Figure 20 shows the occurrence of the deletion according to the present specification.

[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, but other known materials and methods similar or equivalent to those described herein can be used.

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

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

[0043] When 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 this description, the term "mutant mtDNA" or "abnormal mtDNA" is understood to mean an mtDNA molecule having 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 religated mtDNA molecule, which includes the re-ligation or splicing of nucleotides of the remaining mtDNA genomic sequence following the removal of the deletion. As further discussed herein, a deletion event typically results in the creation of two new sequence fragments consisting of a parental sequence corresponding to the religated mtDNA molecule after deletion removal and a deletion sequence corresponding to the deleted portion. Generally, the parental sequence is longer than the deletion sequence. Often, and as discussed above, both the long and short fragments religate to form what are known as the major and minor subcircles, respectively. As understood, both subcircles 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 subcircle.

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

[0047] As used herein, the term "detect" is understood to mean determining or identifying and / or measuring or quantifying the presence of a particular feature in a biological sample. In one aspect, the term "detect" is used herein to refer to the identification of mitochondrial DNA (mtDNA) sequences, more particularly mtDNA having deletions. 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 comprises an amino acid sequence resulting from the translation of the religated mtDNA following a deletion event. Such mtDNA may contain wild-type mtDNA, or abnormal or deleted sequences.

[0048] As used herein, the term "diagnose" is understood to mean identifying a disease state or stage of a disease, or determining a high or increased probability of the presence of a disease state or stage of a disease. For example, with respect to the present description, when an mtDNA molecule or fusion transcript described herein is detected, a higher probability of the presence of a stage or state of endometriosis is considered to exist, or is "diagnosed". It is understood that the actual or clinical diagnosis of this stage or state is made by a clinician upon examination of a biopsy sample or other such means. Thus, in some instances, the terms "detect" and "diagnose" may be used interchangeably herein.

[0049] As used herein, the term "biological sample" is understood to refer to a tissue or body fluid containing cells or nucleic acids from which the molecule of interest can be obtained. A biological sample can be used directly as obtained from the source or can first be subjected to a pretreatment to alter the characteristics of the sample. In one embodiment, the biological sample is blood, particularly circulating blood, and as used herein, the term "blood" is understood 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 the biological sample. It is understood that a blood sample for the purposes of this description can be taken from any source on the body of the subject. This includes, but is not limited to, blood taken from a venous source by syringe etc., collection of a menstrual fluid sample, or capillary blood such as blood taken by finger prick. Using the presently described methods with circulating blood (including blood derivatives as above) provides an effective means of detecting the presence of endometriosis in individuals suspected of having such a condition without subjecting them to an unnecessarily painful and risky invasive procedure. As noted above, in situations where the presently described methods suggest the presence of endometriosis, a diagnosis still requires a clinical evaluation and possibly laparoscopy / surgery or analysis of a biopsy sample. Thus, in one embodiment, particularly when using circulating blood (or one or more of its derivatives as above) as the biological sample, it is understood that the presently described methods can be performed on a subpopulation of patients including those individuals having one or more signs suggesting the presence of endometriosis. It is also understood that the presently described methods can be performed on members of the general population as an initial step in screening for endometriosis. In other words, the presently described methods can be performed on subjects without signs (i.e., individuals not presenting with symptoms).

[0050] As used herein, the terms "mitochondrial fusion transcript" or "fusion transcript" refer to RNA transcripts produced as a result of the transcription of mtDNA sequences.

[0051] As used herein, the term "variant" refers to a nucleic acid sequence that is different from a naturally occurring sequence but retains its essential or functional properties. In one aspect, the term "variant" refers to a sequence that varies with respect to a wild-type sequence. Generally, in the case of mtDNA, variants are overall closely similar and, in many regions, are identical to the selected mtDNA sequence. In the context of this description, a variant may include at least one nucleotide of the nucleotide at the junction point of a spliced gene and may further include one or more nucleotides adjacent thereto. In one aspect, the variant 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 or its complementary strand described herein.

[0052] As used herein, the phrase "substantially similar" refers to nucleic acids that are functionally the same but differ in each nucleic acid sequence. In one aspect, two sequences that are substantially similar to each other may be referred to as "variants". Thus, two nucleic acid molecules may be considered to be substantially similar if one or more nucleotide differences between their respective nucleic acid sequences do not change their functional properties or the functional properties of any polypeptide encoded by such nucleic acids. As will be appreciated, due to the degeneracy of the genetic code, a change in a base pair results in no change in the encoded amino acid sequence.

[0053] The term "substantial complementarity" refers to a sufficiently high degree of complementarity between the nucleotide sequences of nucleic acid molecules, which allows hybridization between them, but is not necessarily 100% complementary. For example, a primer or probe having substantial complementarity to a target sequence may have 80% - 99% sequence identity to the target sequence. In one aspect, 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 portion of a given mitochondrial genome sequence or a nucleic acid sequence that is the complementary strand thereto. In one aspect, such a "portion" includes at least two nucleotides that include the junction point of a spliced gene and may further include one or more nucleotides adjacent thereto. That is, the portion includes a recombined or religated DNA sequence after removal of a deletion. Fragments described herein are at least about 150 nucleotides (nt) in length, 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. Specific minimum nucleotide lengths are listed above, but it is understood that 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 when described herein.

[0055] In the context of the length of a sequence, as used herein, the term "about" includes a value that is larger or smaller by several (5, 4, 3, 2, or 1) nucleotides at either or both ends of a specifically recited value.

[0056] As used herein, the terms "probe" or "primer" refer to an oligonucleotide molecule that forms a double-stranded structure with a target nucleic acid or "hybridizes" to the target nucleic acid due to complementarity between a portion of the nucleotide sequence of the target molecule and at least a portion of the nucleotide sequence of the probe / primer. The target nucleic acid molecule can, in some cases, be a fragment of a naturally occurring nucleic acid molecule. The probes described herein can 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 are known to those of skill in the art. The probes herein can also be referred to as hybridization probes. The probes and primers described herein can be of any length as will be understood by those of skill in the art. By way of example only, the probes and primers currently described can 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 one 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 understood by those of skill in the art, a probe can have a longer nucleotide length than a primer. Thus, in some cases, the probes described herein can 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 any particular probe or primer length.

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

[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 rather is understood to mean "at least one" or "one or more".

[0060] As described herein, the inventors have identified novel mtDNA deletions. The novel mtDNA deletions are, in one aspect, associated with endometriosis and, therefore, constitute an accurate diagnostic marker for such a condition. The inventors have also identified novel mtDNA fusion transcripts. The novel mtDNA fusion transcripts are, in one aspect, associated with endometriosis. Both of these aspects are discussed further below. Translation products resulting from the fusion transcripts are also encompassed by this description.

[0061] In one aspect, the description of the present invention relates to the hypothesis of the inventors that endometrial cells shed into menstrual fluid during menstruation would have the same genetic profile as endometrial-like cells present in ectopic and / or orthotopic endometrial lesions. The inventors further hypothesized that mitochondrial deletions and fusion transcripts may be present in endometrial-like cells present in ectopic and / or orthotopic endometrial lesions, using findings obtained from mapping large-scale deletions of the human mitochondrial genome, observations of the high frequency of these deletions, and evidence in other disease types of transcriptionally activated mutant mtDNA molecules.

[0062] To test these hypotheses, 268 mitochondrial fusion transcripts were selected based on predicted direct and indirect repeats across the entire mitochondrial genome and screened for use as biomarkers of endometriosis. A number of mtDNA deletions and corresponding fusion transcripts were identified by the inventors as being particularly useful in distinguishing samples having endometriosis from samples not having endometriosis. These deletions and fusion transcripts are discussed further below. These mtDNA molecules produce fusion sequences having open reading frames (ORFs) 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 described above, mtDNA mutations generally involve deletions of portions of the mtDNA wild-type sequence. This description is based on the association of specific mtDNA mutations, specifically deletions of the mtDNA genomic sequence, with endometriosis.

[0064] According to the present description, to determine a candidate genomic sequence, junction points resulting from sequence deletions were first identified. The sequence deletions were mainly identified by direct or indirect repeat elements adjacent to the sequences deleted at the 5' and 3' ends. When a portion of nucleotides is removed from the genome and then the remaining genome is ligated, new junction points are created.

[0065] In the identification of junction points, to identify spliced genes, the nucleotides of the genes adjacent to the junction points were determined. Typically, a spliced gene contains a start codon from a first gene and a stop codon of a second gene and can be expressed as a continuous transcript, i.e., retaining the reading frame from the beginning to the end of both spliced genes. There may also be a possibility of using another start codon or stop codon contained within the gene sequence.

[0066] Large deletions in the mitochondrial genome often result in two products from the mutation process. These products are the result of the religation of both portions of the mtDNA genome of 1) a short sequence (in one aspect, which may correspond to the deleted mtDNA sequence) and 2) a long sequence (in one aspect, which may correspond 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 and long sequences religate to form what are known as small subcircles and large subcircles, respectively. If the small component has an insufficient number of nucleotides, religation is not possible, and in that case, the mutation process results in only large subcircles. As discussed herein, both large and small subcircles can be identified, thereby enabling both molecules to be used for detecting, diagnosing, and / or observing endometriosis.

[0067] ((1.2) Fusion transcript) Large-scale rearrangement mutations in the mitochondrial genome result in the generation of fusion transcripts. Therefore, it was predicted that mtDNA rearrangements associated with endometriosis would result in fusion transcripts also associated with endometriosis. Accordingly, the use of mtDNA encoding such transcripts and probes directed thereto for the diagnosis and monitoring of endometriosis is provided herein.

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

[0069] ((1.3) Translation product) Based on the fusion transcripts described herein, this description also provides the amino acid sequences of the putative proteins resulting from the translation of the subject fusion transcripts, i.e., "fusion proteins". This description also provides the translation products of at least a portion of the fusion transcript, specifically a portion containing the transcribed fusion site or junction point of the 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] Assays for 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 assay (ELISA) and radioimmunoassay (RIA). 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 can also be produced by recombinant techniques known in the art. Typically, this involves 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 of the present description can be produced against the wild-type or expressed fusion proteins described herein, or antigenic polypeptide fragments thereof, which may be presented to an animal system (e.g., rabbit or mouse) together with a carrier protein (e.g., albumin), or, if it is long enough (at least about 25 amino acids), may be presented without a carrier. Although antibodies are described, it is understood that any other suitable binding agent specific for the identification of the protein may also be used. In any case, the antibody or binding agent can identify the fusion proteins described herein by specifically binding to regions of such proteins that represent or indicate deletions. In one embodiment, the fusion protein has a unique amino acid profile representing the translation of the junction point of the mtDNA molecule (either the major or minor sub-loop) after the deletion event.

[0074] As used herein, the terms "antibody" (Ab) or "monoclonal antibody" (Mab) mean intact molecules, and antibody fragments, or antigen-binding fragments thereof (e.g., Fab and F(ab')2 fragments, etc.), that can specifically bind to or have "specificity" for a mitochondrial fusion protein. Fab and F(ab')2 fragments lack the Fc fragment of the intact antibody, disappear more rapidly from the circulation, and may have less non-specific tissue binding of the intact antibody (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 a polyclonal antiserum with a higher specific activity.

[0076] In related methods, the antibodies of the present description are monoclonal antibodies. Such monoclonal antibodies 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, N.Y., (1981) pp. 563-681). Generally, such procedures involve immunizing an animal (preferably a mouse) with a mitochondrial fusion protein antigen or a cell expressing a mitochondrial fusion protein.

[0077] In one aspect, the present description includes immunological assays that use antibodies or antigen-binding fragments having specificity for the fusion proteins described herein (as described above). Such immunological assays can be facilitated by kits that include the antibody or antigen-binding fragment, along with any other necessary reagents, test strips, materials, instructions, etc.

[0078] (assay) Measuring the level of translation products such as fusion proteins in a biological sample can determine the presence or progression of endometriosis in a subject. Thus, in one aspect, the present description provides a method for predicting, diagnosing or observing endometriosis, which comprises obtaining one or more biological samples, extracting mitochondrial fusion proteins from the samples, and assaying the samples for one or more molecules therein by quantifying the amount of the one or more molecules and comparing the detected amount to a reference value. As will be understood by those skilled in the art, the reference value is based on whether this method is intended to predict, diagnose or observe endometriosis. Thus, the reference value may 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 proteins in samples 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 )). Further methods useful for detecting protein expression include immunoassays such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA).

[0080] In one aspect, the present description provides a method for detecting, diagnosing or observing endometriosis in a mammal, the method comprising assaying a tissue sample derived 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) mtDNA hybridization probes and / or primers that can hybridize to abnormal mtDNA sequences under appropriate hybridization conditions are also described herein. 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., the molecules listed in Table 1 below), or against fragments or variants thereof. For example, the abnormal mtDNA sequences discussed herein can be used to design primers or probes that detect nucleic acid sequences containing the fusion nucleotide sequence of interest. As will be appreciated by those skilled in the art, primers and / or probes that hybridize to these nucleic acid molecules can hybridize under highly stringent hybridization conditions or under less stringent conditions. Such conditions are known to those skilled 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 comprise sequences complementary to at least a portion of abnormal mtDNA that includes the junction point of a spliced gene. As noted above, this “portion” includes at least two nucleotides that remain in the mtDNA genome after deletion removal, thereby creating a junction point. The junction point is specified herein as A:B, where “A” and “B” represent mtDNA genomic nucleotides on opposite sides of the deletion sequence and are adjacent to each other after the remaining sequences are religated. 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 nucleotides involved in 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 can be modified by one or more base pairs while still allowing hybridization to the target sequence. Such a primer or probe is said to have “substantial complementarity” to the target sequence. As noted above, both major and minor subcircles can result after a deletion event, and both such subcircles have respective junction points as defined above once the molecule is religated.

[0084] Further, the description includes, in one embodiment, primers designed to span the deletion junction or junction point A:B in the forward or reverse direction. In another embodiment, one or more primers can 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 being considered 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. A detectable label is a molecule or a molecule that can be detected directly or indirectly, and is selected such that it does not affect the ability of the probe to hybridize to its target sequence. 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 this description include those that can be detected directly, such as radioisotopes, fluorophores, chemiluminophores, enzymes, colloidal particles, fluorescent microparticles, etc. One of ordinary skill in the art will understand that directly detectable labels may require additional components such as substrates, trigger reagents, light, etc. to enable detection of the label. This description also contemplates the use of labels that are detected indirectly.

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

[0088] The probes described herein preferably hybridize to nucleic acid molecules derived from the biological samples described herein, thereby enabling the described methods. Accordingly, in one aspect, a hybridization probe for use in the detection of endometriosis is provided, the probe being complementary or substantially complementary to at least a portion of the abnormal mtDNA molecules described herein or a portion of the deletion sequences derived from the mtDNA genome.

[0089] ((2.2) Fusion Transcript Probes and Primers) Once a fusion transcript is characterized, primers or probes can be developed to target the transcript in a biological sample. 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 transcript, and detection techniques (e.g., QuantiGene(™) 2.0 by Panomics(™)) can be used to detect the presence of the transcript in the sample. Primers and probes can be generated directly against the exemplary fusion transcripts described herein or fragments or variants thereof. For example, the sequences described herein (e.g., the 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, 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 contains at least two nucleotides complementary to the expressed junction point and may further contain one or more additional adjacent complementary nucleotides. In this regard, the description encompasses any suitable targeting mechanism for selecting fusion transcripts that uses nucleotides involved in 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 the present 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 "at least 20 nt in length" is intended to include, for example, 20 or more consecutive bases complementary to the mtDNA sequence of the present invention. Of course, larger probes (e.g., 50, 150, 500, 600, 2000 nucleotides) are preferred. As noted 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 the detection of endometriosis are provided, wherein the one or more probes and / or primers are complementary, or substantially complementary, to at least a portion of the mitochondrial fusion transcripts described herein.

[0093] <(3.0) Assays for Detecting mtDNA Deletions, Fusion Transcripts, and Their Protein Products> As described above, the present description provides mitochondrial DNA biomarkers useful in detecting, diagnosing, and / or observing endometriosis in a subject using a biological sample derived from the subject. Specifically, such biological samples are non-invasively collected menstrual fluid, circulating blood, and / or tissue (such as biopsy tissue). The present description thus provides, in one aspect, a menstrual fluid- or blood-based test that enables early and accurate detection of endometriosis, thereby preventing unnecessary initial and repeated surgical procedures. Accordingly, the methods described herein reduce the need for unnecessary laparoscopic procedures when endometriosis is suspected but not detected. The method also aids in determining whether endometriosis has recurred by enabling the 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, stage, or progression of endometriosis in a subject. This description thus provides a method for detecting, diagnosing, and / or observing endometriosis in a subject, the method comprising assaying a biological sample derived 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-endometriosis" (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 suffering from endometriosis. Such a sample may be described herein as a "known endometriosis" (i.e., "related") biological sample. If the control includes a value or amount from a non-endometriosis source, it may be described herein as a "non-endometriosis amount". In other aspects described herein, the control may include a reference value for another specimen from the same biological sample. In some cases, as further described herein, the amount of abnormal mtDNA can first be normalized relative to the amount of nuclear DNA (e.g., that encoding one or more housekeeping genes (e.g., a gene encoding rRNA)) taken from the same subject. In one aspect, the nuclear DNA sequence used can encode 18S rRNA. The normalized value of the mtDNA can then be compared to a threshold value. When detecting or diagnosing endometriosis, an increase in the amount of abnormal mtDNA in the subject indicates endometriosis.When observing endometriosis, biological samples can be taken from a subject over time and compared over a predetermined period. An increase over time in the amount of one or more abnormal mtDNAs described herein indicates the onset, recurrence, or progression of endometriosis in a subject.

[0095] The presently described methods also include assaying a biological sample for a panel of abnormal mtDNA markers described herein, such panels containing mtDNA markers from two or more subjects. For example, such a panel can contain 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the presently described mtDNA markers.

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

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

[0098] In another embodiment, the present specification provides a method for detecting endometriosis, and the assay comprises: a) performing a hybridization reaction using at least one probe as described herein to enable 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 abnormal 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 with at least one known reference value, where - if the reference value includes the amount of mtDNA not associated with endometriosis, the higher the amount of abnormal mtDNA in the sample, the more likely endometriosis is present; or - if the reference value includes the amount of mtDNA associated with endometriosis, the lower the amount of abnormal mtDNA in the sample, the less likely endometriosis is present.

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

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

[0101] ((3.2) Measurement of fusion transcripts) Measuring the levels of the mitochondrial fusion transcripts described herein in a biological sample can also determine the presence, stage or progression of endometriosis in a subject. Accordingly, there is provided a method for detecting, diagnosing and / or observing endometriosis, the method comprising 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 include 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 be related 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 the subject over time.

[0102] In one aspect, the methods described herein include 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] Accordingly, in one aspect, there is provided a method for detecting endometriosis in a mammal, the method comprising hybridizing a biological sample (e.g., blood, menstrual fluid, or tissue) from the mammal with at least one hybridization probe having a nucleic acid sequence complementary to at least a portion of a mitochondrial fusion transcript to assay for the presence of at least one fusion transcript described herein, the portion including a fusion junction in the mitochondrial fusion transcript.

[0104] In another aspect, a method is provided that includes assaying a biological sample derived from a mammal by hybridizing the sample to at least two primers. As noted above, at least one of the primers can have a sequence that permits hybridization to a portion of a fusion transcript that includes a fusion junction. In other aspects, the primer can have a sequence that permits hybridization to a flanking region of the fusion junction.

[0105] In another aspect, the invention provides the method described above, and the assay includes: a) performing a hybridization reaction using at least one of the probes mentioned above, and hybridizing at least one probe 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 the transcript hybridized to at least one probe; and, c) comparing the amount of the mitochondrial fusion transcript in the sample to at least one known reference value, where: - where the reference value includes the amount of mitochondrial fusion transcripts not associated with endometriosis, the higher the amount in the sample, the more likely endometriosis is present; and, - where the reference value includes the amount of mtDNA associated with endometriosis, the lower the amount of abnormal mtDNA in the sample, the less likely endometriosis is present.

[0106] ((3.3) Detection of the Translated Protein) The translation products of the fusion transcripts described herein, the proteins, can be detected using generally known methods such as immunological assays that utilize antibodies or other such specific binding components. Specifically, such components specifically bind to the translated fusion site or junction of the mtDNA.

[0107] <(4.0) Kit> This description encompasses a diagnostic or screening kit for the in vitro detection, diagnosis, and / or observation of endometriosis in a subject. Such a kit preferably includes one or more probes or primers as described herein, optionally in combination with reagents, instructions, tools, and / or containers, etc., if required to perform an assay.

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

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

[0110] In one aspect, this description provides a kit for performing an in vitro assay for the purpose of detecting and / or diagnosing endometriosis, which includes the hybridization probes described herein and at least one reagent for performing the assay.

[0111] In one aspect, the kit described herein includes 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 noted above, in one aspect, a portion of the sequence to which the probe hybridizes includes a junction point or fusion junction in the mtDNA or fusion transcript. In one aspect, the kit may include one or more probes adapted to hybridize to one or more control sequences.

[0112] In another aspect, the kit described herein includes a pair of primers (e.g., a forward primer and a reverse primer) for amplifying at least a portion of the abnormal mtDNA described herein or at least a portion of the mitochondrial RNA fusion transcript described herein. In one aspect, 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 aspect, 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 aspect, the kit may include 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> mtDNA mutations (or abnormal mtDNA) and fusion transcripts that have been found to be useful in the methods claimed herein are described below. Predicted translation products are also provided and are thought to be useful for the same reasons. Probe and primer sequences useful for detecting the subject's mtDNA and fusion transcripts are also provided below.

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

[0115] [Table 1] TIFF2025087867000003.tif142170

[0116] In Table 1, "Deletion ID" is a reference number for identifying mtDNA deletions among the screened ones. "SEQ ID NO." refers to the nucleotide sequence identifier assigned to the mtDNA deletion of the subject in this specification. "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. "Position of deletion" identifies a portion of each sequence removed from the parental mtDNA molecule. In the next column, "Spliced 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. "Position in mtDNA" identifies the segment of the mtDNA sequence corresponding to the wild-type mtDNA genome (i.e., SEQ ID NO: 1). "Junction site" identifies the position of the junction point of the mutant mtDNA following the removal of the deletion (based on the wild-type mtDNA genome, SEQ ID NO: 1). Thus, as an example, for deletion ID number 4 (SEQ ID NO: 3) having a "Junction site" of 7586-7992 / 15730-15887, the deleted mtDNA segment contains nucleotides 7993 to 15729. In such a case, once the abnormal mtDNA is circularized again, it contains junctions at nucleotides 7992 and 15730. The part within the parentheses in this column identifies the position of the splice in each SEQ ID NO. The last column identifies the flanking repeat sequences of the deletion. The repeats shown within the angle brackets are removed together with the deletions shown in the third column.

[0117] As shown in Table 1, one of the flanking repeat sequences is removed along with the deletion sequence, resulting in forming a part of the deletion sequence. However, the other repeat sequence may instead be included with the deletion. This deletion mechanism is shown in Figure 20, which shows the parental mtDNA molecule 10, where 12 and 20 represent opposite ends of the mtDNA molecule, 16 represents the deletion, or the deletion sequence (such as those listed in the third column of Table 1), and 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, resulting in forming a part of the deletion 16. Thus, once the remaining parental mtDNA is religated, it contains segment 12-18-20 or segment 12-14-10, as shown in Figure 20. As described above, although it is stated that an entire repeat is included in the deletion sequence, it is possible that only one or both parts of the repeat are included in the deletion.

[0118] The mutant mtDNA sequences according to this description can include any modifications that result 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 the presently described mtDNA mutations can be selected from any techniques known to those skilled in the art. For example, the analysis of mtDNA can include target selection by DNA branching, sequencing of mtDNA, amplification of mtDNA by PCR, Southern, Northern, Western, Southwestern blot hybridization, denaturing HPLC, hybridization to a 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. This description encompasses the use of these variants or fragments of the sequences for diagnosing and / or observing 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 were detected and found to be useful in the detection, diagnosis, and / or observation of endometriosis, as shown in the Examples.

[0122] [Table 2]

[0123] In Table 2, "Transcript number" is the identification number assigned to the fusion transcript and corresponds to the mtDNA deletion ID number in Table 1. "mtDNA deletion sequence number" is the mtDNA deletion sequence identifier in Table 1. "Transcript sequence number" is the sequence identifier of the fusion transcript of the subject. "Fusion transcript name" indicates the "FUS" designation, and A:B represents the junction point between the last mitochondrial nucleotide of the first spliced gene and the first mitochondrial nucleotide of the second spliced gene. "Flanking gene" indicates the spliced gene resulting from the deletion. "Deletion junction" indicates the position of the junction point of the mtDNA molecule after 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. Generally, this involves the 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) Table 3 provides 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.

[0128] [Table 3]

[0129] 〔Example〕 The following examples are given to further illustrate aspects of the present invention. The examples are in no way intended to limit the scope of the specification.

[0130] (Example 1: Screening for large-scale fusion transcripts in endometrial tissue) 268 probes corresponding to fusion transcripts were screened by endometrial tissue samples for evidence of differential expression relative to control samples in samples obtained from endometriosis patients. The screening methods and results are described hereinafter.

[0131] Generation of probe library 268 probes were identified using a unique program to find nucleotide base pair repeats. This program identified over 16,000 possible deletions based on direct and indirect repeat elements adjacent to sequences to be deleted at the 5' and 3' ends. The selection of the 268 probes was based on the criterion that a minimum of 8 base pair repeats was required; there could also be deletions less than 8 base pair repeats. As an example, the repeat of deletion 16 is 3 bp.

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

[0133]

Table 4

[0134] Using the samples listed in Table 4, tissue homogenates were prepared using the QuantiGene™ Sample Processing Kit for “fresh or frozen animal tissue”. For each sample, four pieces of frozen endometrial tissue (each approximately 100 mg) were cut and weighed prior to addition to 6 mL of lysis solution containing 60 μL of proteinase K. Samples were homogenized using a Qiagen tissue disruption probe and incubated overnight at 65 °C. The homogenates were then clarified by two centrifugations at 16,000 × g for 15 minutes. The supernatants were saved and used as templates for the subsequent branched DNA assay. Alternatively, DNA was extracted from tissue homogenates or directly from fresh frozen tissue according to the tissue protocol using the Qiagen QiaAmp™ DNA Mini Kit. The DNA was then quantified using a Nanodrop™ spectrophotometer and normalized for subsequent use in qPCR reactions.

[0135] Mitochondrial DNA deletions and the resulting fusion transcripts can be detected using one of many molecular techniques. Here, branched DNA and quantitative PCR techniques were used to detect fusion transcripts and parental abnormal mtDNA molecules, respectively.

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

[0137] Next, the homogenate was added to the capture plates and incubated overnight at 55 °C to allow probe-template hybridization. Following a series of washing and hybridization steps, a chemiluminescent substrate was added. Degradation of the alkaline phosphatase bound to the probe-template hybrid generates a luminescence signal reported as relative light units (RLU). Each capture plate was read twice in duplicate, and the RLU was measured with a Promega Glomax™ luminometer. The RLU values were analyzed bioinformatically. The two plate readings and three values were averaged, provided that they had a coefficient of variation (CV; i.e., the ratio of the standard deviation to the mean) of 15%. Also, it was determined whether the RLU value exceeded the background for a given probe. Specifically, the lower limit of quantification, LOQ (LOQ = mean RLU of the probe background + 10 standard deviations of the background mean) was calculated and subtracted from the sample RLU. Subsequently, the sample RLU values were converted to log2 or log10 values to facilitate the analysis. Finally, for any given probe, the sample RLU was standardized against the housekeeping (HK) RLU by subtracting or dividing the sample RLU from itself. The standardized results of 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 FIGS. 2A - 2J and summarized in Table 5 below.

[0138]

Table 5

[0139] Table 5 shows the standardized average RLU values (Log2LOQProbe - Log2LOQHK23) for control and endometriosis positive (“Endo.Pos.”) tissue samples (corresponding to the scatter plots shown in FIGS. 2A - 2J). The average difference and significance of the difference between the two tissue groups are given. The average copy numbers of the given fusion transcripts (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). The purified DNA extracts were standardized against a concentration of 0.25 ng / μL using nuclease - free ultrapure water. The qPCR reaction was carried out at room temperature in the dark 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 specific to 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 having at least one nucleotide pair that forms a junction point after removal of the deletion. Thus, in one aspect, a junction primer can overlap with both nucleotides forming the junction point or only one of said nucleotides. In some cases, as shown in the tables below, more than two sets of primers were used. The reaction was made up to a final volume of 25 μL using PCR - grade H 2 O. The reaction mixture was cycled in either a Chromo 4™ (Biorad) or Opticon 2™ (MJ Research) real - time PCR cycler.

[0141]

Table 6

[0142]

Table 7

[0143] Results and Discussion As shown above, approximately 268 fusion transcripts were screened during 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, elevated levels in endometrial tissue 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) were found to be associated with endometriosis. The presence of each transcript was determined by assaying each probe having a nucleotide sequence at least complementary to the portion of the transcript having the junction point.

[0144] The scatter plots and performance of all fusion transcript probes are shown in FIGS. 2A-2J and Table 5. FIG. 3 shows the positions of the fusion transcripts throughout the mitochondrial genome, the gene positions within the genome, and the positions of the 10 mtDNA fusion transcripts of the present invention (i.e., “probes” or “targets”) within the genome (indicated by lines spanning the length of each deletion). Probes corresponding to the aforementioned fusion transcripts were tested against three endometriosis-positive and four endometriosis-negative endometrial samples (see Table 1). For each sample, the RLU values were normalized to 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 was concluded that fusion transcripts 1, 4, 14, 16, 120, 122, 193, 400, 516, and 586 (i.e., transcripts having the 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, in this study, it was found that elevated levels of the target transcripts in endometrial tissue samples are highly correlated with endometriosis. Detection of the target fusion transcripts can be achieved using the probes identified above, which have nucleotide sequences that are at least sufficiently complementary to at least a portion of the nucleotide sequences of each of the fusion transcripts. This portion includes the 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 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 for the detection of endometriosis. Such deletions can be identified by identifying the junction points of the parental mtDNA (i.e., the re-circularized major sub-loop) after re-circularization. The junction points can be identified using a probe having a nucleotide sequence that is at least substantially complementary to at least a portion of the mtDNA nucleotide sequence containing the junction point (such that the probe hybridizes to each mtDNA). The 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 having the junction point. Alternatively, the primers can include pairs 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 sequences (i.e., the re-circularized minor sub-loop) after re-circularization.

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

[0149] Accordingly, as described herein, a method for the detection of endometriosis is provided, where the method includes 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 the respective mitochondrial fusion transcripts and their abnormal parental mtDNA molecules. In particular, the probes described herein are designed to be at least substantially complementary to a fusion transcript encoding a transcribed junction point corresponding to the recombined (or circularized) mtDNA. The primers described herein are preferably designed such that one of the primer pairs has a nucleotide sequence complementary to a junction point of abnormal mtDNA that has been circularized following removal of the deletions described herein. It will also be understood that other primer pairs (where one of the primer pairs is at least substantially complementary to a junction point of a deleted sequence that has been circularized, or where the primer pair is at least substantially complementary to an mtDNA nucleotide sequence adjacent to the junction point) can be designed.

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

[0151]

Table 8

[0152] As described above, the 1.2 kb deletion refers to the deletion of nucleotides 9087 to 10312 based on the wild-type mtDNA genome (SEQ ID NO: 1). Therefore, such a deletion results in a large sub-loop (having a junction between nucleotides 9086 and 10313 when re-circularized) having bases 0 to 9086 and 10313 to 16568. Similarly, the 3.7 kb deletion refers to the deletion of nucleotides 9189 to 12905, resulting in a large sub-loop (having a junction between nucleotides 9188 and 12906 when re-circularized) having bases 0 to 9188 and 12906 to 16568.

[0153] As described above, the re-circularization of the large sub-loop has been discussed. However, if the re-circularized small sub-loop has a unique junction point as taught, the re-circularization of such a small sub-loop is also appropriate. In this study, small sub-loops corresponding to the 1.2 kb and 3.7 kb deletions were identified during the process of sequencing the samples. Therefore, the findings in this example can be extended to the detection of small sub-loops resulting from the deletions described herein.

[0154] Method Participants and Specimen Collection This study utilized unprocessed, unidentified clinical specimens collected from pre-registered patients as part of the EndOx study at the Oxford Endometriosis Centre, John Radcliffe Hospital, University of Oxford. Briefly, specimens were collected from women scheduled to undergo laparoscopy for suspected endometriosis due to pelvic pain (symptomatic) or tubal ligation (asymptomatic). Study participants were women aged 18 years or older (pre-menopausal) who were confirmed to be non-pregnant. All specimens were obtained under a research protocol that had received appropriate ethical approval from the National Research Ethics Service (Oxfordshire REC A, 09 / H0604 / 58). All clinical specimens were anonymized to protect the identity of the source patients. This trial was designed, conducted, and reported in accordance with the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Good Clinical Practice guidelines and the ethical principles defined in the applicable national regulations and the Declaration of Helsinki. All patients provided written informed consent prior to participation.

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

[0156] Patient Population / Study Cohort The clinical specimens used in this study were classified as asymptomatic controls, symptomatic controls without surgically confirmed endometriosis, or cases of surgically confirmed endometriosis. Asymptomatic controls were defined as specimens collected from patients scheduled to undergo tubal ligation who had no clinical suspicion of endometriosis and were surgically confirmed to be free of endometriosis. Symptomatic controls were defined as specimens collected from patients with pain or other symptoms (excluding infertility) who were clinically suspected of having endometriosis but in whom no endometriosis lesions were visualized by laparoscopy performed by an experienced gynecological 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, deep endometriosis) and rASRM stage (stages I through 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 the QIAamp 96 QIAcubeHT (trademark) extraction kit (Qiagen, Crawley, UK) automated on the QIAcube HT (trademark) system (Qiagen, Crawley, UK). The extracted DNA was eluted in 200 μL of AE buffer.

[0159] Real-time qPCR for mtDNA deletions Amplification was performed in a 20-μL reaction volume using a 96-well microplate (Bio-Rad, Hemel Hempstead, UK). Each well contained 5 μL of non-normalized DNA template, 1× SYBR Green (registered trademark) master mix, and 250 nM of each primer. The primers used in the reaction are shown in Table 9.

[0160]

Table 9

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

[0162] Standardization of Real-time qPCR by 18S rRNA The amount of target amplicon was standardized using the nuclear DNA gene of 18S rRNA. The amplification reaction was performed as a 20 μL reaction volume in a 96-well microplate. Each well contained 5 μL of non-standardized DNA template, 1× SYBR Green® master mix, and 200 nM of each primer. The amplification and fluorescence of SYBR Green® I were analyzed using a Chromo4 real-time PCR detection system. The amplification conditions were 3 minutes at 95 °C, followed by 40 cycles of 30 seconds at 95 °C, 30 seconds at 64.5 °C, and 30 seconds at 72 °C. After amplification, melting curve analysis from 70 °C to 90 °C was performed, reading every 0.5 °C.

[0163] Quality control The quantitative cycle (Cq) was calculated using the CFX Manager software regression model (Bio-Rad, Hemel Hempstead, UK). The Cq of each deleted amplicon was normalized to the Cq of the 18S rRNA gene amplicon, which is a nuclear target of other copies. All samples were amplified in triplicate on separate plates, and were considered qualified if at least two of the three replicates were within a range of 1.5 Cq and the melting point (Tm) was not contrary to the target amplification product when present (Tm of deletion 81°C ± 2°C, Tm of 18S rRNA 82°C ± 2°C).

[0164] Two control samples without template were processed together with each batch of DNA extraction and were confirmed negative for the amplification of both the deleted target and the 18S rRNA gene. Two control reaction solutions without template were placed for each PCR plate and were confirmed negative for the amplification of both the deleted target and the 18S rRNA gene. The specificity of the deletion primers was evaluated using rho 0 cellular DNA (to detect mitochondrial pseudogene amplification), DNA from buccal swabs of healthy males, and DNA extracted from the parental cell line of rho 0 (before mitochondrial deletion).

[0165] For the first round of standard PCR reaction, DNA extracted from patients with confirmed endometriosis was subjected to whole-genome amplification using the Repli-G™ Mitochondrial DNA Kit (Qiagen) to ensure sufficient amount of DNA at this stage.

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

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

[0167] Statistical analysis Without performing a formal calculation of the sample size, the number of clinical specimens used was determined to be sufficient to meet the study objectives.

[0168] For qPCR, the target was amplified in triplicate from all specimens, and the mean Cq value was calculated. We determined the normalized deletion value (ΔCq) by quantifying the deletion amplicon relative to the 18S rRNA reference amplicon. For receiver operating characteristic (ROC) curves and descriptive statistics, statistical analysis was performed using GraphPad Prism™ 5.0 (GraphPad Software Inc, La Jolla, CA, USA). 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 mean, standard deviation (SD), and range for continuous variables. The means of two groups were compared using Student's t-test and Mann-Whitney U-test for parametric and non-parametric distributions, respectively. The correlation between two variables was evaluated using the Pearson correlation coefficient (r). For the presence of endometriosis, ROC curves were generated for all except the 6.5 kb deletion. The area under the ROC curve (AUC), as well as sensitivity and specificity at the selected cut-off (described below), were calculated with 95% confidence intervals (CI). For all tests, a p-value < 0.05 was considered statistically significant.

[0169] Results Patient population and clinical specimens Demographic and clinical characteristics of the patients who provided the clinical specimens used for the 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 / specimens; SD = standard deviation. (1) Mean (standard deviation) is shown; mean values and standard deviations were calculated for patients from whom age at specimen collection was obtained. (2) Status of patients within 3 months of specimen collection. (3) Menstrual status of patients at specimen collection.

[0172] The clinical and demographic characteristics of the patients and specimens used for the evaluation of the 1.2 kb and 3.7 kb differed and only included those samples with qPCR results paired for each deletion that met the 18S rRNA and previously described acceptance criteria.

[0173] 1.2 kb deletion cohort 171 specimens were used for the evaluation of the 1.2 kb deletion. The mean (SD) age of the patients who provided the specimens was 34.2 (6.8) years. The mean age was similar in the control and case groups, with mean (SD) ages of 36.6 (6.9) years and 33.7 (6.7) years, respectively, and there was no statistically significant difference (p = 0.113). Of the 171 patient specimens used for the evaluation, 116 (67.8%) patients had no hormone therapy within 3 months before specimen collection, 48 (28.1%) had hormone therapy within 3 months before specimen collection, and 7 (4.1%) reported unclear. Phase data of the menstrual cycle were calculated using the last menstrual period (LMP) before blood collection in relation to the patient's normal cycle length. 24 (14.0%) patients reported amenorrhea - 19 of them were on hormone administration, 12 (7.0%) had irregular menstruation, 31 (18.1%) were in the menstrual phase (between 1 - 5 days from the first day of LMP), 44 (25.7%) were in the follicular phase (5 - 14 days from LMP), and 60 (35.1%) were in the luteal phase + extended menstrual phase (15 days or more from LMP).

[0174] The control group included a total of 28 specimens; 18 specimens (64.3%) were collected from symptomatic patients (who presented symptoms consistent with endometriosis other than infertility and were confirmed to have no surgery for this disease), and 10 specimens (35.7%) were collected from asymptomatic patients scheduled for tubal ligation. The test group included 143 specimens collected from patients with three disease subtypes (peritoneal, ovarian, deep infiltrating [DI] endometriosis) classified into four stages (rASRM I-IV). 49 specimens (34.3%) were collected from women with peritoneal endometriosis, 45 specimens (31.5%) were collected from women with ovarian endometriosis, and 49 specimens (34.3%) were collected from women with deep endometriosis. 63 specimens (44.1%) were from patients with stage I disease, 21 specimens (14.7%) were from stage II, 29 specimens (20.3%) were from stage III, and 28 specimens (18.6%) were from stage IV. 2 specimens (1.4%) had an unknown disease stage.

[0175] 3.7 kb deletion cohort 181 specimens were used for the 3.7 kb deletion assessment. The mean (SD) age of the patients who provided the specimens was 34.4 (6.9) years. The mean age was similar in the control group and the case group, with mean (SD) ages of 37.2 (6.8) years and 33.8 (6.8) years, respectively, and there was no statistically significant difference (p = 0.166). 119 (65.7%) of the patients had no hormone therapy within 3 months before specimen collection, 55 (30.4%) had hormone therapy within 3 months before specimen collection, and 7 (3.9%) reported unclear. 26 (14.4%) of the patients reported amenorrhea, 15 (8.3%) had irregular menstruation, 31 (17.1%) were in the menstrual period (1-5 days), 44 (24.3%) were in the follicular phase (5-14 days), and 65 (35.9%) were in the luteal phase + extended menstrual period (more than 15 days).

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

[0177] mtDNA Deletion and Preliminary Assessment - Standard PCR Based on sequence composition, the presence of adjacent repeat positions within the major arc of the mitochondrial genome where relatively many deletions have been reported

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

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

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

[0180] Preliminary evaluation with clinical specimens As a preliminary evaluation of the remaining six candidates, deletions were evaluated using a set of 55 clinical specimens; 46 specimens from patients with confirmed endometriosis and 9 specimens from symptomatic control patients. After the initial QPCR test, 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 co-amplification of numts), and the 7.7 kb deletion amplified only at low stringency annealing temperatures (meaning that mispriming events were more likely to occur). These candidate deletions did not meet the assay requirements as planned here, but could still exist as biomarkers that could 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 at a copy number sufficient to facilitate reliable and simple detection. This 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 endometriosis disease cases (combined across all subtypes and stages). The AUC (95% CI) for the 1.2 kb deletion was 0.8116 (0.6178 - 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 - 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 specimens in the evaluation set; PCR = polymerase chain reaction.

[0184] Diagnostic accuracy of the 1.2 kb and 3.7 kb deletions To further fully evaluate the 1.2 kb and 3.7 kb deletions as clinically applicable biomarkers for endometriosis, the ability of these deletions to distinguish between symptomatic controls and all types of combined endometriosis, between the three subtypes of endometriosis and between the four stages of endometriosis was determined using a larger clinical specimen population (Table 10). Valid paired results (both target and 18S gene amplification) were obtained for 171 specimens with the 1.2 kb deletion and 181 specimens with the 3.7 kb 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 presenting with symptoms of endometriosis with surgical confirmation of disease status as an outcome). Importantly, the 1.2 kb deletion and the 3.7 kb deletion did not detect a difference between symptomatic and asymptomatic control specimens at p = 0.462 and p = 0.878, respectively.

[0185] Symptomatic control vs. all diseases Similar to the preliminary analysis using 55 clinical specimens, both the 1.2 kb deletion and the 3.7 kb deletion accurately distinguished between symptomatic control specimens and endometrial disease specimens (combined peritoneal, ovarian, and deep endometriosis specimens). 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). The coordinates of the receiver operating (ROC) curve were examined and the threshold, i.e., the cut-off, was selected to optimize sensitivity. Applying a threshold of -4.43 for the distinction 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 deletion and the 3.7 kb deletion improved the diagnostic accuracy between all symptomatic control groups and all endometriosis (AUC 0.827 (0.722 - 0.931), and AUC 0.882 (0.784 - 0.980) between the symptomatic control group and stage I / II disease (data not shown).

[0188] Disease by subtype - 1.2 kb deletion An important feature of any diagnostic adjunct for endometriosis is the ability to accurately detect all disease subtypes. We evaluated the ability of the 1.2 kb deletion to distinguish between symptomatic control specimens and specimens taken 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 differences in the amount of the normalized 1.2 kb deletion between symptomatic controls were 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 (the AUC (95% CI) values were 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 the detection of deep endometriosis). Overall, these data indicate that the 1.2 kb deletion was able to accurately distinguish specimens collected from symptomatic controls from those of peritoneal, ovarian, and deep endometriosis patients. When applying a threshold of -4.430 for the distinction between symptomatic controls and peritoneal endometriosis tp using the 1.2 kb deletion, the sensitivity and specificity values were 81.8% and 72.2% respectively. At a threshold of -4.675, the sensitivity and specificity of the 1.2 kb deletion in the distinction between symptomatic controls and ovarian endometriosis were 75.6% and 72.2% respectively. At a threshold of -4.350, the sensitivity and specificity of the 1.2 kb deletion in the distinction between symptomatic controls and deep endometriosis were 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 differences in amplicon amounts among symptomatic controls were statistically significant for peritoneal endometriosis (p<0.0001), ovarian endometriosis (p<0.0001), and deep endometriosis (p = 0.0072). The diagnostic accuracies of the 3.7-kb deletion for detecting each of the three disease subtypes are shown in Figures 6B - 6D (AUC (95%CI) values were 0.8978 (0.8131 - 0.9824) for detecting peritoneal endometriosis, p<0.0001, 0.8158 (0.7003 - 0.9313) for detecting ovarian endometriosis, p<0.0001, and 0.7110 (0.5746 - 0.8475) for detecting deep endometriosis, p = 0.0071). Collectively, these data indicate that the 3.7-kb deletion was able to accurately distinguish between symptomatic controls and samples taken from women with peritoneal, ovarian, and deep endometriosis. Applying a threshold of 8.805 for differentiating symptomatic controls and peritoneal endometriosis using the 3.7-kb deletion resulted in 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 for differentiating symptomatic controls and ovarian endometriosis were 80.9% and 68.4%, respectively. At a threshold of 11.01, the sensitivity and specificity of the 3.7-kb deletion for differentiating symptomatic controls and deep endometriosis were 80.0% and 52.6%, respectively (Table 12).

[0191] Disease by stage - 1.2-kb deletion Another important feature of the biomarker for endometriosis is its ability to detect both the low and high stages of the disease. Next, the ability of the 1.2 kb deletion to distinguish between symptomatic control specimens and specimens of confirmed low (I / II) or high (III / IV) stages of the disease was evaluated. The distribution of the 1.2 kb deletion for stage I / II and stage III / IV diseases 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 differences between symptomatic controls were statistically significant for the stage I / II (p < 0.0001) and stage III / IV (p = 0.001) disease groups. The difference between stage 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 stage I / II detection and 0.7661 (0.6398 - 0.8924), p = 0.0007 for stage III / IV detection. Thus, the 1.2 kb deletion was able to 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 were 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 were 80.7% and 72.2% respectively (Table 12).

[0193] Disease - 3.7 kb deletion by stage The distribution of the 3.7 kb deletion for stage I / II and III / IV diseases is shown in Figure 8A. The mean (SD) ΔCt values were 11.12 (2.239) in symptomatic controls, 8.243 (2.156) in stage I / II, and 8.112 (2.14) in stage III / IV. The differences between symptomatic controls were statistically significant for the stage I / II (p < 0.0001) and stage 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) for stage I / II detection, p < 0.0001, and 0.7591 (0.6354 - 0.8837) for stage III / IV detection, p = 0.0007. The difference between stage I / II and III / IV was statistically significant for the 3.7 kb deletion (p = 0.016). These data indicate 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 were 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 were 84.5% and 52.6%, respectively.

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

[0195]

Table 13

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

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

[0198] For the 1.2 kb deletion, it was determined that there was no correlation with disease detection and patient age; the correlation coefficient (r) was r = 0.030 (p = 0.698). Also, no correlation was observed between disease detection and the specimen age for each collection year (r = 0.072, p = 0.353). When stratified by hormonal status (patients who had received hormonal therapy or had not received hormonal therapy within 3 months before specimen collection), the difference in disease detection was not statistically significant (p = 0.120). When patients were stratified by menstrual phase (amenorrhea, irregular menstruation, menstruation, follicular phase, luteal phase + enlarged menstruation), there was no statistically significant difference in disease detection due to 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 specimen age (r = 0.0628; p = 0.4009), nor was it significantly affected by hormonal therapy (p = 0.195). Detection of endometriosis by the 3.7 kb deletion was significantly correlated with the menstrual phase (p = 0.036) and clearly appeared with a difference of 1.72 in the difference in detection between patients without a period and those in the follicular phase (p = 0.026). Overall, these data indicate that the accuracy of the 1.2 kb and these clinically relevant variables are not significantly affected, and that the accuracy of the 3.7 kb deletion is slightly affected by the phase of menstruation.

[0200] Discussion Endometriosis is a disease that is very common in women of reproductive age, associated with a large economic burden, and results in a significant decline 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 most current gold standard in diagnosis is thorough visualization by laparoscopy, ideally followed by histological confirmation of suspicious lesions [5,15]. The diagnostic value of this method is largely unclear because the available objective data are scarce. The use of laparoscopy is potentially inaccurate and is thought in part to range from 60% to 85% according to reports, even when combined with the accuracy of histological confirmation [48-51]. The standard diagnostic method can become even more complex in cases where the disease itself presents atypically or in the early stages of the disease that are not easily visualized and missed 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 an accurate biomarker test would provide the evidence needed to support this treatment. Therefore, it is clearly necessary to improve the current standards so that more consistent results can be provided, especially early in the course of the disease.

[0201] In this study, two novel mtDNA deletions were identified and evaluated as potential biomarkers for endometriosis. Assays targeting 1.2 kb and 3.7 kb deletions meet the criteria for a sound diagnostic test, utilize minimally invasive specimens, and, if successfully translated to clinical use, may potentially help shorten the time to diagnosis associated with current diagnostic practices and provide opportunities for medical intervention prior to surgical procedures. After setting the diagnostic threshold (as described above), the sensitivity of the 1.2 kb deletion assay was 81.8% and the specificity was 72.2%. The diagnostic performance of the 3.7 kb deletion assay was similar, with sensitivity and specificity of 85.1% and 57.9%, respectively. Thus, diagnostic assays based on either of these deletions may complement current standard medical care. Of particular importance is the diagnostic accuracy of these deletions for early-stage disease, as late-stage disease is more readily detected in current clinical practice using ultrasound. In primary care facilities, a positive test result may support the initiation of primary treatment for endometriosis, such as oral contraceptives, or serve as a trigger for referral to a specialist. Among women presenting with dysmenorrhea, an estimated 10% have secondary dysmenorrhea, mostly due to endometriosis

[52] . In this population, the 1.2 kb and 3.7 kb deletions effectively rule out endometriosis with a negative predictive value (NPV) of 97%. In secondary care facilities, a positive test result may serve as a guide for deciding whether to initiate treatment with secondary medications, such as gonadotropin-releasing hormone antagonists, or proceed with laparoscopic surgery. Importantly, in the former facilities, the risk associated with false positive results is less critical, so the diagnostic cut-off value can be selected to maximize the sensitivity of the test, while in the latter facilities, different diagnostic cut-off values that maximize specificity and minimize the exposure of disease-free women to the risks associated with these interventions may be beneficial.

[0202] In addition to diagnostic accuracy, the ability of these two biomarkers to detect endometriosis was not correlated with the patient's age, the age of the specimen, or the 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 specimen collection. Further studies are needed to confirm whether this correlation with the menstrual phase exists in a larger patient cohort or is an artifact of the sample size used in this study. We demonstrated that both the 1.2 kb and 3.7 kb deletions accurately detected all subtypes and stages of the disease. In contrast to current diagnostic standards, which include visualization during surgical procedures and potential resection for histological confirmation, the mtDNA deletion-based assay requires only a blood specimen and can provide objective results before or instead of surgical intervention. Thus, if successfully translated to clinical use, the mtDNA-based assay could reduce diagnostic delays

[16] and provide treatable outcomes earlier in the disease course than has been possible heretofore.

[0203] From a practical perspective, the use of a blood-based biomarker assay has several advantages that effectively improve current standard medical care. Specimens are easy and inexpensive to obtain via venipuncture, and there is a low likelihood of developing a disease state associated with the collection. Blood specimens can be easily obtained in the office of a primary care physician or clinic and do not require the 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 enrichment procedures, and sufficient DNA can be recovered from a standard blood specimen, so a low failure rate of the test can be expected. The assay uses cost-effective PCR-based methods widely used in clinical laboratories, the assay is quantitative, and the results are easily interpretable (i.e., the test results are either above or below a clear diagnostic cut-off corresponding to either a positive or negative result). Finally, the lack or minimal correlation with the menstrual phase ensures that the sample collection requirements are simplified and that the timing of menstruation does not need to be considered when scheduling a venipuncture.

[0204] An important element in the management of chronic diseases is understanding disease epidemiology. Due to the relatively complex diagnostic methods and symptoms overlapping with other gynecological diseases, the epidemiology of endometriosis is not well characterized and varies by patient population and geographical location [1, 2, 4, 6]. The emergence of molecular assays as described herein has made additional data more readily available and can help fill some of the gaps in our understanding of endometriosis epidemiology. Importantly, in this study, widely available and standardized methods were utilized for specimen collection and processing. This allows for more direct comparison of test results across different studies and patient populations [41 - 44, 53].

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

[0206] Based on the criteria shown here, the 1.2 kb and 3.7 kb mtDNA deletions are associated with endometriosis; however, further studies are needed to understand what mechanistic role this mitochondrial genome region plays in the development of endometriosis.

[0207] Limitations of this study include the use of patients who reported their hormone and menstrual status (which may be less accurate than employing specific data measurements for the study). This data is encouraged but requires replication and validation in a larger independent dataset. This study is ongoing.

[0208] Summary The following is a summary of the studies described above: · Endometriosis is a major 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 due to the various clinical pictures and symptoms that overlap with other gynecological diseases. As a result, the definitive diagnosis can be delayed by up to 10 years, leading to high mortality and a low quality of life for affected patients.

[0210] · Thus, there is a clear need for reliable and rapid diagnostic aids that can produce treatable outcomes early in the course of the disease.

[0211] · Test specimens were collected from women scheduled to undergo laparoscopy due to pelvic pain (symptomatic) or tubal ligation (asymptomatic). Study participants were women 18 years of age or older (premenopausal) who were confirmed to be not pregnant.

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

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

[0214] ·The 1.2 kb and 3.7 kb deletions accurately detected endometriosis, including all subtypes and stages, and the detection was not correlated with the age of the patient or specimen or hormonal therapy. The 3.7 kb deletion was significantly correlated with the menstrual phase, limited to two phases.

[0215] ·Biomarkers derived from the mitochondrial genome containing the 1.2 kb and 3.7 kb deletions described herein provide a largely untapped and promising means to achieve the goal in the search for diagnostic markers for endometriosis that can be effectively translated for clinical use.

[0216] ·Based on minimally invasive specimens, assays based on these markers can positively impact the diagnostic outlook for endometriosis by shortening the delay in diagnosis and providing actionable, objective, and rapid test results.

[0217] Conclusion Biomarkers obtained from the mitochondrial genome, particularly the 1.2 kb and 3.7 kb deletions described herein, provide a largely unexplored and promising means to achieve the goal of identifying diagnostic markers for endometriosis that can be effectively translated for clinical use. Based on minimally invasive specimens, assays based on these markers may be recognized to accurately diagnose endometriosis in patient-derived blood samples. Thus, this description provides a rapid, accurate, and effective means for diagnosing endometriosis, thereby shortening delays in obtaining a diagnosis and managing the necessary treatment protocol. This description enables the subject diagnosis to be performed on one or more of the mtDNA deletions (including either the large or small subgenomic) and any resulting fusion transcripts. The same conclusion can be extended to any translation products resulting from the fusion transcripts.

[0218] (Example 3: Identification of an 8.7 kb mtDNA deletion for endometriosis detection) In this study, an 8.7 kb deletion (deletion ID number 2767) was identified 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 method used for this identification is described in more detail in Example 4. We directly detected this biomarker in the tissue lesions of endometriosis using both qPCR and NGS. Based on the 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 the observations in endometrial tissue, the 8.7 kb deletion was selected for evaluation. The 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 genes between NADH dehydrogenase subunits 2 to 5. The first round of standard (quantitative) PCR and visualization after gel electrophoresis were used to pre-limit each of the deletion targets, and each of the candidates was used to determine whether (i) it was detectable; (ii) it had a sufficient copy number for reliable detection; (iii) it had the expected amplicon size; and (iv) it was specific and did not amplify with nuclear pseudogenes or produce non-specific amplification products.

[0221] The deletion was successfully detected in circulating plasma and further evaluated by qPCR to determine whether the target was detectable in rho 0 cells using a 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 three replicates).

[0222] Further studies of this deletion are described in Example 4.

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

[0224] Methods Diagnostic accuracy - Participants and sample collection This was a case-control study using residual plasma specimens collected prospectively from women aged 18 years or older (pre-menopausal), not pregnant (symptomatic controls and endometriosis cases) or having had tubal ligation (asymptomatic controls) who were scheduled to undergo laparoscopy because of suspected endometriosis due to pelvic pain. This trial was part of the EndOx trial at the John Radcliffe Hospital, University of Oxford, Oxford Endometriosis Centre, UK.

[0225] Sample and data collection, anonymization and processing were as previously reported [26, 41 - 44, 57]. The conduct of the trial, 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 The samples taken were classified as either control samples or case samples. The control group consisted of a) asymptomatic control samples taken from participants who had undergone tubal ligation as scheduled without clinical suspicion of endometriosis and had been surgically confirmed not to have endometriosis, and b) symptomatic control samples taken from participants with pain or other symptoms (excluding infertility) associated with clinical suspicion of endometriosis but without endometriosis lesions visible by laparoscopy by experienced gynaecological surgeons.

[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) stages (I: minimal; II: mild; III: moderate; IV: severe)

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

[0228] Disease specificity - Participants and sample collection Endometriosis cases and controls based on diagnostic accuracy assessment were utilized for disease specificity evaluation and compared with the remaining plasma samples obtained from OBIO (El Segundo, USA) and the Ontario Tumour 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 at 2500×g for 10 minutes at 4°C within 1 hour of collection. 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 the QIAamp® 96 QIAcube® HT extraction kit (Qiagen, Crawley, UK) automated by the QIAcube® HT system (Qiagen, Crawley, UK), and the extracted DNA was eluted with buffer AE (200 μL).

[0231] qPCR for mtDNA deletion and normalization of qPCR using 18s rRNA For both real-time polymerase chain reaction (qPCR) procedures, we performed amplification in a 20 μL reaction volume using a 96-well microplate (Bio-Rad, Hemel Hempstead, UK) containing non-normalized 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) by SYBR Green I fluorescence, the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hemel Hempstead, UK) was used.

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

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

[56] . Briefly, the quantification cycle (Cq) was calculated and the Cq of the deletion amplicon was normalized to the Cq of the 18s rRNA gene amplicon, a multi-copy nuclear target. We amplified all samples in triplicate on separate plates. Two control samples without template were processed with each batch of DNA extraction and confirmed 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 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 Formal sample size calculations were not performed, and the number of clinical specimens used was determined to be sufficient to meet the study objectives. For qPCR, the target was amplified in triplicate from all specimens, and the mean Cq value was calculated. We determined the standardized deletion value (ΔCq) by quantifying the deletion amplicon relative to the 18S rRNA reference amplicon. For ROC, descriptive statistics, correlation, and significance testing, statistical analysis was performed using GraphPad Prism™ 5.0 (GraphPad Software Inc, La Jolla, CA, USA). We summarized clinical characteristics using counts and percentages for categorical data and mean, standard deviation (SD), and range for continuous variables. We summarized clinical characteristics using counts and percentages for categorical data and mean values, standard deviation (SD), and range for continuous variables. The means of two groups were compared using Student's t-test and Mann-Whitney U-test for parametric and non-parametric distributions, respectively. The correlation between two variables was evaluated using Spearman correlation (r) or Mann Whitney's U-test or Kruskal-Wallis test. For the presence of endometriosis, an ROC curve was generated for all except the 6.5 kb deletion. The area under the ROC curve (AUC), as well as sensitivity and specificity at the selected cut-off, were calculated with 95% confidence intervals (CI). For all tests, a p-value < 0.05 was considered statistically significant.

[0238] Results Study Population and Clinical Specimens Demographic and clinical characteristics of the participants who provided specimens are summarized in Table 16.

[0239]

Table 16

[0240] Abbreviations: N = number of participants / samples; SD = standard deviation. (1) Mean (SD) is shown; mean and SD were calculated for participants presenting age at sample collection. (2) Status of participants within 3 months after sample collection. (3) Menstrual status of participants at 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. Most of the participants (121; 66.5%) had not received hormone therapy within 3 months before sample collection. Most of the participants who reported not being menstruating were taking hormones (20 / 26; 76.9%).

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

[0243] Preliminary evaluation of 8.7 kb mtDNA deletion - standard PCR As described above in Example 3, we had 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 our own data mining software. As described above, the deletion was successfully detected in circulating plasma, and further evaluation by qPCR was performed to determine whether the target was detectable in rho 0 cells using a more sensitive qPCR.

[0244] Diagnostic accuracy of the 8.7 kb deletion Since the 8.7 kb deletion was successfully detected in circulating plasma and endometriosis lesions, we examined whether the 8.7 kb deletion could be distinguished between symptomatic controls and all endometriosis; among the three subtypes; and between the revised American Society for Reproductive Medicine (r-ASRM) classification stages in plasma from a larger population of clinical specimens. We performed an analysis mainly using specimens from symptomatic controls and participants with confirmed disease to more accurately reflect the clinically important patient population (i.e., all those presenting with endometriosis symptoms). We also measured the frequency of deletions in asymptomatic control specimens, but no difference in the 8.7 kb deletion was detected between symptomatic and asymptomatic control specimens (p = 0.681).

[0245] Comparison of symptomatic controls vs. all diseases Using the 8.7 kb assay, symptomatic controls and all endometriosis specimens could be well distinguished. 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 (a threshold of 6.650 distinguished between symptomatic controls and all subtypes / stages of endometriosis and showed acceptable sensitivity and specificity values (Table 17)) to optimize sensitivity.

[0246] [Table 17]

[0247] Detection of diseases 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 specimens from symptomatic controls from those from patients with peritoneal endometriosis, ovarian endometriosis, and deep infiltrating (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 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 accurately distinguished specimens from symptomatic controls and disease subtypes: the area under the curve (AUC; 95% confidence interval [CI]) was 0.8882 (0.8043 - 0.9722; p < 0.0001) for the detection of peritoneal disease, 0.7766 (0.6572 - 0.8960; p = 0.0008) for the ovary, 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 between symptomatic controls versus peritoneal endometriosis, ovarian disease, and DI disease (Table 17).

[0249] Disease detection at each stage of r - ASRM Endometriosis cases were classified into two stages groups: r-ASRM stages I / II and III / IV, and it was examined whether both low-stage and high-stage diseases could be accurately identified using an 8.7 kb deletion. The 8.7 kb deletion assay distinguished specimens from symptomatic controls from specimens from patients with low stage (stage I / II) and high stage (stage III / IV) (Figs. 14A - 14C), and the mean (SD) ΔCt values were 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] The diagnostic accuracy evaluated by the receiver operating characteristic curve was highest for stage I / II: AUC 0.8361 (0.7426 - 0.9295; p < 0.0001) compared to stage III / IV: AUC 0.7465 (0.6232 - 0.8697; p = 0.0021). At a threshold of 6.65, the 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, hormone therapy, and menstrual phase. We found no correlation between the ΔCt value and patient age (p = 0.749) or between the ΔCt value and specimen age by collection age (p = 0.222) (Table 3). Similarly, no statistical significant difference in the ΔCt value was seen 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 the detection of endometriosis. Kruskal Wallis was used to examine the effect of the menstrual cycle on the detection of endometriosis.

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

[0255]

Table 19

[0256] Data based on Table 19 are shown in Figure 15. Figure 15 shows the standardized 8.7 kb deletion distributions for specimens from participants with endometrial cancer, ovarian cancer, breast cancer, symptomatic controls, and peritoneal, ovarian, or deep infiltrating endometriosis.

[0257] Discussion In this study, we demonstrated the utility of measuring 8.7 kb deletion biomarker levels in plasma samples as a promising assay for detecting endometriosis. Our assay meets the sound criteria for a diagnostic test, with the best performance seen in the peritoneal subtype of endometriosis and at low stages (both of which are frequently encountered in primary care settings), utilizes minimally invasive specimens from blood, and accurately detects all subtypes and stages of the disease. When translated to clinical use, this assay could shorten the time to diagnosis and enable medical intervention prior to surgical procedures. The specimens are easy and inexpensive to obtain via venipuncture and have a low likelihood of associated disease states.

[0258] According to its particularly good diagnostic accuracy for particularly low-stage and peritoneal diseases, the 8.7 kb deletion assay can complement current standard medical care in particularly low-stage and peritoneal diseases, which cannot be reliably detected by imaging methods unlike ovarian and deeply invasive 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 taken in primary care without the need for a dedicated surgical space or instrument. The high copy number of mtDNA means that standard DNA extraction methods can be used without an enrichment procedure. Furthermore, the sufficient amount of DNA recovered from standard blood specimens does not initially show a high failure rate for the test. The real-time PCR-based technique is used in clinical laboratories that produce easily interpretable and quantitative results.

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

[0260] The current complex diagnostic process, combined with symptoms overlapping with other gynecological diseases, means that the epidemiology of endometriosis is not well characterized, which is an important factor in the successful management of any condition [1,2,4,6]. The emergence of molecular assays and new biomarkers will provide more accessible and additional data to help deepen the understanding of the epidemiology of endometriosis. Importantly, our study uses a widely available and standardized method for collecting and processing samples, enabling a more direct comparison of test results across studies and patient populations [41-44,53].

[0261] Conclusion The above-described assay using an 8.7 kb deletion biomarker derived from mitochondria is a minimally invasive method based on blood samples for diagnosing endometriosis that can be used in both primary and secondary medical facilities. The relatively simple and patient-friendly approach provided by this assay shortens the time to diagnosis, thereby improving the management of the debilitated state described herein and thereby improving the quality of life of the patient.

[0262] (Example 5: Identification of a 4.8 kb mtDNA deletion for endometriosis detection) A study similar to the study described above was conducted to identify the correlation between the frequency of a 4.8 kb mtDNA deletion (deletion ID8590) and endometriosis. The method of Example 4 was followed for this analysis. The primers used in this study are shown in Table 20.

[0263] [Table 20]

[0264] Data showing the usefulness of the 4.8 kb deletion in the detection of endometriosis are provided in Table 21 and shown in FIGS. 12-14.

[0265] [Table 21]

[0266] The above description includes references to specific embodiments, but various modifications of those embodiments will be apparent to those skilled in the art. All examples given in this specification are included for illustrative purposes only and are in no way intended to be limiting. All drawings given in this specification are for the sole purpose of illustrating various aspects of the description and are in no way intended to be limiting or to represent size. The scope of the claims appended hereto should not be limited by the preferred embodiments described above, but should be given the broadest interpretation consistent with the specification as a whole. The disclosures of all documents cited in this specification are hereby incorporated by reference in their entirety.

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[0268]

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Claims

[Claim 1] 1. A method for identifying an abnormal mitochondrial DNA (mtDNA) molecule having a deletion in a biological sample from a mammalian subject, comprising: the deletion comprises the 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; Once recircularized, the mtDNA comprises a junction point.

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