{"paper_id":"d245eac2-3698-4ca6-b331-3449cb0d937f","body_text":"Endometriosis (EM) is a chronic oestrogen-dependent disease characterised by the ectopic presence of active endometrium outside the uterine cavity, as in the myometrium (i.e., adenomyosis), ovaries, uterosacral ligaments, bladder, and pelvic peritoneum, and even out of the pelvis as well [ 1 ]. It is a common gynaecological disorder, affecting approximately 10–15% of women of reproductive age [ 1 , 2 ]; however, considering that some affected patients may remain paucisymptomatic with advanced EM and, conversely, even extremely distressing symptoms may be overlooked, the disease is probably underdiagnosed, and the current estimates do not reflect the true prevalence of the disorder [ 3 ]. The clinical presentation of EM can be subtle [ 1 ]. Additionally, affected women often experience infertility [ 4 ].\nTo date, there are several hypotheses to explain EM aetiology [ 5 ]. The most widely accepted is the “retrograde menstruation” theory, recently updated with the “stem cell” one [ 6 ]. However, it still does not explain (i) why the retrograde menstruation, which happens in the majority of women, leads to EM only in a small percentage of them, and (ii) the occurrence of EM in females with Rokitansky syndrome [ 7 ] and in males [ 8 ]. Therefore, the biological and molecular pathways involved in the etiopathogenesis of this disorder are still unclear.\nEM is recognised as a multifactorial disorder, in which both genetic and environmental factors play significant roles [ 9 ]. Concerning the environmental factors, pollution exposure and diet appear to be the main ones involved. For instance, dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)) has been widely implicated in EM pathogenesis [ 10 , 11 ], while regarding diet, phytoestrogens and saturated fats have been linked with this disease [ 12 ].\nAside from the environmental factors, several studies have highlighted the contribution of genetics to the aetiology of this complex disease. The first formal genetic study on EM was conducted in 1971 by Simpson and colleagues, who demonstrated the familial clustering of this disorder [ 13 ]. Since then, several additional family studies have been performed, showing a higher concordance for monozygotic tweens than dizygotic couples, and wide population-based studies have estimated that the heritability of EM is approximately 50%, suggesting that it may follow a polygenic inheritance pattern [ 14 ]. In recent years, Genome-Wide Association Studies (GWASs) have proven to be an effective tool to identify EM-associated genes [ 15 , 16 ]; many of them appear to be involved in sex-steroid-hormone signalling,  WNT  signalling, cell adhesion and migration, cell growth, and inflammation-related pathways. Although GWASs have been extremely useful in identifying many genes potentially involved in the etiopathogenesis of EM, their findings are able to explain less than 5% of the phenotypic variance; additionally, the exact causative link between the identification of a susceptibility locus and the underlying molecular pathways that leads to disease development remains, in many cases, unclear [ 16 ]. In this light, new approaches are needed to fill the current knowledge gap regarding EM pathophysiology to allow for the identification of novel molecular targets that can be implemented as diagnostic, prognostic, and therapeutic biomarkers.\nTo this purpose, in this study, we performed whole-exome sequencing (WES) analysis on a highly selected cohort of 80 deeply characterised patients with the final goals of identifying rare variants in 46 known EM-associated genes and discovering new potentially causative ones. Furthermore, in order to deepen the complex polygenic nature of EM, a comprehensive evaluation of the analysed genes was performed, testing the hypothesis that the burden of genes harbouring rare and damaging variants is higher in EM patients than in a control cohort.\n\nWritten informed consent to participate in the study and for the collection of biological samples for research purposes was obtained from all participants. The study was conducted in accordance with the Helsinki Declaration and was approved by the Ethics Committee of the Friuli-Venezia Giulia region (Italy) (Prot. n. 47846 dd. 27.12.2022).\nA total of 80 adult women with confirmed surgical or clinical diagnoses of EM were recruited at the I.R.C.C.S. “Burlo Garofolo” Hospital in Trieste (Italy). Confirmation of the EM diagnosis was based on the following: (1) both visual inspection with histological confirmation for the patients that underwent surgery (i.e., laparoscopy or laparotomy); (2) imaging techniques performed by expert operators, for the remaining subjects, such as transvaginal or transrectal and transabdominal ultrasound according to the International Deep Endometriosis Analysis (IDEA) group consensus [ 17 ], and/or magnetic resonance imaging (MRI) according to the European Society of Urogenital Radiology (ESUR) guidelines [ 18 ]. The EM severity was staged considering the revised American Society for Reproductive Medicine (rASRM) classification [ 19 ].\nAt enrolment, all patients underwent a deep clinical evaluation. In particular, detailed information regarding demographic data (i.e., age, anthropometric measurements), past and familial medical history, and gynaecological anamnesis (i.e., age of menarche, EM diagnosis, number of pregnancies, infertility diagnosis) were collected. Furthermore, a careful evaluation of the most common EM-associated symptoms (i.e., ovulation, pre-menstrual and post-menstrual pain, dysmenorrhea, dyspareunia, dyschezia, dysuria) before medical therapy or surgery was performed for each patient.\nSpecifically, the presence of the abovementioned pain was registered as a dichotomous variable (“yes”/“no”), and the intensity of pain was rated with a 0–10-point Numerical Rating Scale (NRS) (i.e., 0 represents “no pain at all” and 10 represents “the worst imaginable pain”) [ 20 ].\nFor each patient, a peripheral whole-blood sample was collected for genomic DNA extraction. The protocol for genomic DNA extraction was performed as already described in Spedicati et al. [ 21 ].\nWES was carried out on an Illumina NextSeq 550 instrument (Illumina Inc., San Diego, CA, USA) with the Twist Exome 2.0 plus Comprehensive Exome Spike-in kit (Twist Bioscience, South San Francisco, CA, USA), according to the manufacturer’s protocol. The WES protocol and secondary and tertiary analyses were carried out as already reported in Spedicati et al. [ 21 ].\nTwo different phases of WES data analysis were performed. Firstly, a candidate gene approach was carried out, focusing the WES data analysis on a list of EM-associated genes; secondly, an unbiased approach was conducted to detect variants within novel candidate genes. The complete list of the analysed genes is reported in  Table S1 .\nAs regards the candidate gene approach, a list of 46 genes was created based on a literature review, according to the following criteria: (i) each gene had to be described in association with EM in at least two published papers; (ii) only the most recent papers (i.e., published between 2011 and 2023) were considered.\nTo perform variant selection within the analysed genes and novel candidates, the following criteria were applied: Variants with a quality score < 20, Variant Allele Frequency < 30, or called in off-target regions were excluded; A Minor Allele Frequency (MAF) cut-off of 0.1% was considered. The variant frequency was verified both in NCBI dbSNP ( https://www.ncbi.nlm.nih.gov/snp/ , accessed on 30 April 2023) and gnomAD ( https://gnomad.broadinstitute.org/ , accessed on 30 April 2023); The effect of the genetic variants was evaluated with  in silico  prediction tools, such as PolyPhen-2 (tolerated for scores < 0.5, damaging for scores ≥ 0.5) [ 22 ], SIFT [ 23 ], PaPI (tolerated for scores ≤ 0.5, damaging for scores > 0.5) [ 24 ], DANN (tolerated for scores ≤ 0.9, damaging for scores > 0.9) [ 25 ], the dbscSNV score (tolerated for scores ≤ 0.9, damaging for scores > 0.9) [ 26 ], and SpliceAI (tolerated for scores < 0.5, probably damaging for scores ranging from 0.5 to 0.8, damaging for scores ≥ 0.8) [ 27 ]; SNVs leading to synonymous aminoacidic substitutions not predicted as damaging, not affecting splicing, or highly conserved residues were excluded.\nVariants with a quality score < 20, Variant Allele Frequency < 30, or called in off-target regions were excluded;\nA Minor Allele Frequency (MAF) cut-off of 0.1% was considered. The variant frequency was verified both in NCBI dbSNP ( https://www.ncbi.nlm.nih.gov/snp/ , accessed on 30 April 2023) and gnomAD ( https://gnomad.broadinstitute.org/ , accessed on 30 April 2023);\nThe effect of the genetic variants was evaluated with  in silico  prediction tools, such as PolyPhen-2 (tolerated for scores < 0.5, damaging for scores ≥ 0.5) [ 22 ], SIFT [ 23 ], PaPI (tolerated for scores ≤ 0.5, damaging for scores > 0.5) [ 24 ], DANN (tolerated for scores ≤ 0.9, damaging for scores > 0.9) [ 25 ], the dbscSNV score (tolerated for scores ≤ 0.9, damaging for scores > 0.9) [ 26 ], and SpliceAI (tolerated for scores < 0.5, probably damaging for scores ranging from 0.5 to 0.8, damaging for scores ≥ 0.8) [ 27 ];\nSNVs leading to synonymous aminoacidic substitutions not predicted as damaging, not affecting splicing, or highly conserved residues were excluded.\nTo interpret the effect of the selected variants, the genetic intolerance profile for the protein domains was analysed with the MetaDome web server [ 28 ].\nFinally, the correlation between each patient’s variants and clinical phenotype was examined, evaluating the related literature, in order to identify possible relevant genotype–phenotype correlations. All selected variants were confirmed via Sanger sequencing.\nOne hundred and five healthy women recruited during routine gynaecological visits to I.R.C.C.S. “Burlo Garofolo” (Trieste, Italy) were included in this study as a control cohort. This control group was carefully selected according to the following criteria: (1) age over 18 years, (2) absence of clinical and/or surgical diagnosis of EM, and (3) no reports of infertility issues. WES data of these subjects were already available as an in-house database and were analysed following the criteria described in this paragraph.\nThe “burden of genes” was calculated for each individual of the EM and control cohorts. In particular, for each participant, the burden of genes was defined as the total number of genes, considering variants within the 46 selected genes and the novel candidates identified, in which at least one rare and damaging variant was identified after the WES analysis and variant selection. A Wilcoxon two-sample rank test was performed to compare the burden of genes’ distribution between the cases and controls. The statistical significance was set to a  p -value < 0.05. The analysis was performed with R version 4.1.2 (R Foundation for Statistical Computing, Vienna, Austria).\nThe complete workflow of this study is reported in  Figure 1 .\n\nA cohort of 80 EM patients aged between 20 and 57 years were enrolled at I.R.C.C.S. “Burlo Garofolo” Hospital (Trieste, Italy). In particular, 59/80 (74%) patients were diagnosed with EM stage III–IV, and 95% of them underwent surgery. Furthermore, 53% of the EM patients had full-term pregnancies, and 21% received infertility diagnoses. Forty-five percent of patients were currently undergoing a medical therapy (i.e., progestins, combined oestrogen–progestins, or hormone-releasing intrauterine devices), while only 16% took only anti-inflammatory drugs to control EM-associated symptoms. Notably, only 5.8% of patients suffered from dysuria, while most of them (64%) reported dysmenorrhea, with a mean pain intensity rating of 6.8 on the NRS. Complete patient demographic data and clinical features are summarised in  Table 1 .\nThe WES analysis was performed in two steps, firstly focusing on the selected list of 46 EM-associated genes, and then on the novel candidate discovery.\nOverall, this strategy allowed for the identification of 63 rare (MAF < 0.1%), predicted, and damaging variants within 21/46 genes and 3 novel candidates ( ABCA13 ,  NEB , and  CSMD1 ) in 50/80 (63%) EM patients. All variants were detected in the heterozygous state.\nComplete WES results are reported in  Table S2  and graphically represented in  Figure 2 .\nIn particular, the WES analysis results revealed the following: A total of 34/80 (43%) patients carried different rare, predicted, and damaging variants within 13 recurrent genes ( FCRL3 ,  LAMA5 ,  SYNE1 ,  SYNE2 ,  GREB1 ,  MAP3K4 ,  C3 ,  MMP3 ,  MMP9 ,  TYK2 ,  VEGFA ,  VEZT ,  RHOJ ); A total of 7/80 (8.8%) patients (i.e., patients 3, 9, 19, 50, 67, 14, 19, 13) carried different private, rare, predicted, and damaging variants within eight single genes ( KAZN ,  IL18 ,  WT1 ,  CYP19A1 ,  IL1A ,  IL2RB ,  LILRB2 ,  ZNF366 ); A total of 19/80 (24%) patients carried different rare, predicted, and damaging variants within three novel candidate genes ( ABCA13 ,  NEB ,  CSMD1 ).\nA total of 34/80 (43%) patients carried different rare, predicted, and damaging variants within 13 recurrent genes ( FCRL3 ,  LAMA5 ,  SYNE1 ,  SYNE2 ,  GREB1 ,  MAP3K4 ,  C3 ,  MMP3 ,  MMP9 ,  TYK2 ,  VEGFA ,  VEZT ,  RHOJ );\nA total of 7/80 (8.8%) patients (i.e., patients 3, 9, 19, 50, 67, 14, 19, 13) carried different private, rare, predicted, and damaging variants within eight single genes ( KAZN ,  IL18 ,  WT1 ,  CYP19A1 ,  IL1A ,  IL2RB ,  LILRB2 ,  ZNF366 );\nA total of 19/80 (24%) patients carried different rare, predicted, and damaging variants within three novel candidate genes ( ABCA13 ,  NEB ,  CSMD1 ).\nFurther, the WES analysis detected the following: (i) the  FCRL3  ( NM_052939.4 ):c.958T>A variant segregated in patient 34 and her sister (patient 35); (ii) the  MAP3K4  ( NM_005922.4 ):c.3590_3598dupCTGCTGCTG variant detected in patient 28 segregated in two other members of her family (patients 63 and 64 (respectively, her mother and aunt)), both diagnosed with EM.\nRegarding the recurrent genes mentioned above ( Table 2 ), the most compelling results regard the  FCRL3 ,  LAMA5 ,  SYNE1 , and  SYNE2  genes. Four out of eighty patients (5.0%) carried different predicted and damaging variants within the  FCRL3  gene. To note, patient 2 had an infertility diagnosis, and patients 34 and 35, two sisters, reported a family history of infertility.\nIn 5/80 (6.3%) of the EM patients, the WES analysis allowed for the identification of different novel, predicted, and highly impacting missense variants within the  LAMA5  gene; all these selected variants belong to domains of the encoded protein predicted as intolerant to missense variation. Three out of five patients (patients 5, 12, and 23) carrying  LAMA5  variants were diagnosed with EM stage III–IV.\nMoreover, in 7/80 (8.8%) patients, different rare, predicted, and damaging variants within two genes belonging to the same family,  SYNE1  and  SYNE2 , were identified. Notably, six out of seven of the patients (patients 16, 23, 26, 40, 45, and 47) carrying variants within these two genes shared a common clinical feature: a severe, EM-associated, and painful symptomatology, often poorly controlled via antalgic and medical therapy.\nIn 7/80 (8.8%) EM patients, the data analysis identified rare, private, predicted, and damaging variants within single genes ( Table 3 ) that, according to the literature, may have a role in regulating the inflammatory response and infertility mechanisms underlying EM pathogenesis. The most enthralling results concern the  IL18 ,  KAZN , and  WT1  genes.\nIn details, patient 3, a 30-year-old woman with EM stage IV, carried a predicted and damaging missense variant within the  IL18  gene. The genetic-intolerance-profile analysis with MetaDome revealed that this variant belongs to a protein domain predicted as highly intolerant to missense variation.\nPatient 9, a 49-year-old woman with EM stage IV, showed a predicted and damaging missense variant within the  KAZN  gene. A careful evaluation of patient 9’s clinical history revealed that this patient had three pregnancies that all ended with spontaneous miscarriages.\nIn patient 19, a predicted and damaging missense variant within the  WT1  gene was detected. Of note, patient 19 had been diagnosed with infertility and reported severe dysmenorrhea, rated eight on the NRS.\nThe WES data analysis carried out with an unbiased approach revealed that 19/80 (24%) EM patients carried rare, predicted, and damaging variants in three novel candidate genes ( ABCA13 ,  NEB ,  CSMD1 ) ( Table 4 ). In particular, 8/80 (10%) of the EM patients carried different predicted and damaging variants within the  ABCA13  gene, 8 other patients within the  NEB  gene, and the remaining 4 within the  CSMD1  gene.\nFrom a genetic perspective, EM is a polygenic disorder determined by the combined effect of multiple genes. In order to evaluate whether a higher number of genes harbouring rare and damaging variants was present in EM patients than in healthy controls, the “burden of genes” was calculated. Specifically, for each individual of the EM and control cohorts, the burden of genes was defined as the total number of genes in which at least one rare and damaging variant was identified ( Table S3 ). The burden of genes’ distribution ranged [0–3] both in the cases and controls. The medians and interquartile ranges were 1.0 (0.0–1.0) in the EM patients and 0.0 (0.0–1.0) in the controls. A Wilcoxon two-sample rank test determined that the burden of genes was statistically significantly higher  (p  = 0.02) in the EM cohort than in the controls.\n\nEM is a chronic, inflammatory, multifactorial disease with a high prevalence in the general population. To date, it is one of the most underdiagnosed and undertreated disorders, with a mean of 8–12 years between the beginning of symptoms and a definitive diagnosis [ 29 ]. Furthermore, EM is characterised by a heavy social impact, as it has detrimental effects on women’s quality of life, fertility, and social relationships [ 30 ]. The current unavailability of rapid and minimally invasive diagnostic tools poses a complexity for clinicians in the diagnostic process of and therapeutic planning for EM. Therefore, there is an urgent clinical need to detangle the complex genetic and molecular mechanisms underlying this disease’s etiopathogenesis.\nSeveral GWASs have been carried out so far to pinpoint novel EM-associated genes. However, although GWASs are a successful strategy to identify genetic variants underlying multifactorial disorders, they present some limitations. Firstly, GWASs select variants that are associated with the disease of interest, rather than a causal mechanism. Secondly, GWASs can detect only relatively common variants widespread in the population. Thirdly, GWASs require a precise phenotypical characterisation to obtain solid and reliable results, which may be less accurate in large-sample cohorts. For this reason, other genetic approaches are needed.\nIn this light, this study took advantage of a combined approach of a detailed clinical characterisation and WES analysis to deepen, for the first time in the literature, the effect of rare variants within a list of highly selected EM-associated genes and novel candidates. This strategy allowed us to perform accurate genotype–phenotype comparisons, unveiling interesting insights into EM’s underlying pathological mechanisms.\nThe WES analysis identified 63 predicted and damaging variants within 21 genes and 3 novel candidates. All these genes belong to several molecular pathways, such as the regulation of the immune response, cellular proliferation and migration, and oestrogen metabolism, all reported to be involved in EM pathogenesis [ 31 ]. This is a particularly relevant result considering that thorough research of the literature revealed that only a few causative variants and genes are currently described in relation to EM [ 32 ].\nThe most compelling results regard the  FCRL3 ,  LAMA5 ,  SYNE1 , and  SYNE2  genes, in which rare, predicted, and damaging variants were detected in more than one patient, and a consistent genotype–phenotype correlation was identified.\nConcerning  FCRL3 , a gene encoding a member of the immunoglobulin receptor family, rare and damaging variants were identified in 4/80 (5.0%) EM patients. Three out of four of these patients reported a diagnosis and/or family history of infertility. According to the literature, this gene has already been linked with an increased risk of EM-associated infertility, irrespective of the disease stage [ 33 , 34 ]. To date, the cause-and-effect relation underlying EM and infertility is still poorly elucidated, and several genes regulating inflammation and angiogenesis are currently being explored as potential etiologic factors; among them,  FCRL3  is a promising candidate. Indeed,  FCRL3 , besides B cells, is also expressed in natural killer (NK) cells and regulatory T cells (Treg), key modulators of the specific immune response against ectopic endometrial lesions and involved in fertility-mechanism modulation [ 35 ]. Previous studies have shown that the increased levels of  FCRL3 -positive Tregs detected in EM patients could be responsible for a reduced immune response that could enable the implantation of endometrial cells and infertility onset [ 36 ]. In this light, an in-depth characterisation of the  FCRL3  gene’s role in relation to infertility mechanisms could pave the way, in the future, for novel strategies for the better early clinical management of patients with infertility issues.\nRegarding  LAMA5 , this gene encodes the alpha-5 Laminin protein, which has been associated with EM stage III–IV and EM-related infertility [ 37 , 38 ]. In particular, high levels of LAMA5 were detected in the eutopic endometria of EM-stage-III–IV patients during the menstrual proliferative phase, and an association of a  LAMA5  SNP (rs2427284) with EM stage III–IV has been demonstrated [ 37 , 38 ]. In accordance with these findings, in our cohort, three out of five patients carrying variants within this gene were diagnosed with EM stage III–IV, giving relevance to the previously described relation. Further, all five identified  LAMA5  missense variants belong to protein domains predicted as intolerant to missense variation. Therefore, it can be hypothesised that these variants impact on the LAMA5 structure and biological function, thereby promoting the adhesion of endometrial cells in ectopic sites. From this perspective, further studies are needed to unveil the relationship between  LAMA5  variants and EM stage III–IV. This could be relevant to gain a deeper understanding of EM progression mechanisms, and to evaluate the potential predictive and prognostic values of rare and damaging variants within this gene.\nFinally, as regards  SYNE1  and  SYNE2 , another relevant genotype–phenotype correlation was identified.  SYNE1  and  SYNE2  are two genes belonging to the same family, encoding, respectively, the Nesprin1 and Nesprin2 proteins, two structural proteins that share a common function of anchoring the nuclear envelope to the actin cytoskeleton. Notably, six out of seven EM patients carrying damaging variants within these genes reported a severe EM-associated symptomatology. Indeed,  SYNE1  has been recently associated with the most common EM-associated pain symptoms (e.g., dysmenorrhea, dyspareunia, severe dyspareunia, and acyclic pelvic pain) and menstrual migraine [ 16 ]. To note, this gene belongs to the same genomic locus of other genes (i.e.,  ESR1  and  CCDC170 ) involved in oestrogen-hormone signalling, a key player in the regulation of the growth of endometriotic lesions, and in the modulation of pain perception [ 15 ]. Concerning  SYNE2 , its expression was found to be dysregulated in the endometria of EM patients [ 39 ] and, currently, there are no available data associating it with EM-related pain. However, considering that this gene belongs to the same gene family as  SYNE1 , it could be speculated that  SYNE2  could play a similar role in EM pathogenesis; hence, further functional characterisations of  SYNE2  variants are needed to elucidate the role of this gene in EM-associated pain perception.\nOverall, deepening the role of  SYNE1  and  SYNE2  variants in relation to EM-associated pain could be fundamental to shed light on the entangled mechanism underlying EM symptom severity and variability. This will pave the way for the identification of novel molecular markers to be implemented, in the future, in clinical practice, thereby allowing for the better clinical management of patients carrying  SYNE1/SYNE2  variants, with the activation of personalised treatment plans to improve patients’ symptomatology and quality of life.\nOf particular interest also are the  IL18 ,  KAZN , and  WT1  genes, in which private variants in single patients were detected and a genotype–phenotype correlation was defined.\nConcerning the  IL18  gene, encoding Interleukin 18, several studies have demonstrated that this gene regulates the immune response in the human endometrium, activating NK cells. A previous study showed that the  IL18  expression levels in the endometria of EM patients are lower compared to healthy women [ 40 ], and this is associated with decreased NK-cell activity, thus allowing endometriotic lesions to escape immune elimination [ 41 ]. Further, according to the literature,  IL18  also plays a role in female fertility maintenance, regulating uterine receptivity and the embryo-implantation phase [ 42 ]. Indeed, previous studies have shown that women with repeated failures of implantation after in vitro fertilisation showed the dysregulation of  IL18  and other cytokines in the endometrium, compared with fertile women [ 42 ]. All these findings suggest that  IL18  could be a relevant player not only in EM pathogenesis, but also in EM-related infertility mechanisms, defining it as a promising molecular target to be implemented, in a long-term perspective, as a predictive and diagnostic biomarker.\nMoreover, the  KAZN  gene, which encodes a desmosomal protein involved in cell adhesion, cytoskeleton organisation, and embryonic tissue morphogenesis, has also been linked with EM and EM-related infertility [ 37 ]. A compelling genotype–phenotype correlation was found in this study, as patient 3, carrying a damaging missense variant within the  KAZN  gene, had three pregnancies, all of which ended with spontaneous miscarriages. According to the literature, variants within the  KAZN  gene have also been associated with infertility and pregnancy- and labour-related complications [ 43 ]. All these findings led to the hypothesis that  KAZN  could play a relevant role in fertility- and pregnancy-related mechanisms. In this perspective, the characterisation of damaging variants within this gene could allow for novel genetic insights into EM-related infertility and pregnancy complications, thereby paving the way for the identification of new candidate markers to be implemented in clinical routine for early diagnostic and preventive strategies.\nFinally, the  WT1  gene encodes a zinc-finger-containing transcription factor that regulates female fertility [ 44 ] and has recently been linked with EM-associated dysmenorrhea [ 16 ]. Further, it has been reported that  WT1  is involved in determining an aberrant increase in aromatase expression and oestrogen synthesis in the eutopic and ectopic endometria of EM patients [ 45 ]. In this cohort, a reasonable genotype–phenotype correlation was detected, as a damaging missense variant within the  WT1  gene was identified in patient 19, diagnosed with EM, EM-related infertility, and severe dysmenorrhea. Considering the strength of the genotype–phenotype correlation identified,  WT1  could be a promising biomarker; indeed, it could be considered in future clinical practice for the definition of the tailored clinical management of patients carrying variants within this gene aimed at (1) reducing the severity of EM-associated dysmenorrhea and (2) implementing ad hoc preventive strategies for fertility maintenance.\nA noteworthy feature of this study is represented by the peculiar enrichment of EM patients carrying variants within three novel candidate genes,  ABCA13 ,  NEB , and  CSMD1 , which were selected considering their potential biological role in relation to EM pathogenesis. In particular, the  ABCA13  gene, encoding a ganglioside transporter [ 46 ], could be a novel candidate to deepen the entangled mechanisms underlying pain processing. Indeed, alterations in ganglioside metabolism are related to neuropathic and inflammatory pain [ 47 ]. Considering also that variants within the  ABCA13  gene are associated with an increased susceptibility to schizophrenia, bipolar, and major depression disorders [ 46 ], and that EM has been associated with depression and a higher incidence of anxiety [ 30 ], this gene could be a novel player to also scrutinise these aspects.\nThe role of  NEB  in EM’s pathogenesis has yet to be clarified. This gene encodes Nebulin, a sarcomere protein that regulates cytoskeletal dynamics [ 48 ]. Considering that, in EM pathogenesis, the dynamic remodelling of cytoskeleton components is involved in the migration of endometriotic lesions,  NEB  could be a novel candidate to be considered in this process. In line with this, literature data report that the  NEB  gene is frequently mutated in stage III endometrial cancer [ 49 ], and other genes of the Nebulin family, (e.g.,  LASP1 ,  LASP2 ) are involved in cytoskeletal-architecture regulation and focal-adhesion organisation [ 48 ].\nFinally,  CSMD1  encodes a regulator of the complement system, the biological role in regulating fertility mechanisms and cellular proliferation [ 50 ] of which would allow for intriguing insights into EM pathogenesis. Only one GWAS in the literature has reported this gene in relation to EM, but the variant identified did not reach genome-wide significance [ 51 ]. Several studies are currently highlighting its role in regulating cellular proliferation, as  CSMD1  inhibition causes increased cellular invasion, motility, and proliferation [ 52 ]. Therefore, it can be hypothesised that variants within  CSMD1  could impact its biological function, thereby promoting the migration and proliferation of endometrial cells in ectopic sites. However, further in vitro and/or in vivo studies are necessary to characterise the role of  CSMD1  in EM onset.\nFinally, in order to deepen the complex genetic architecture of EM, a comprehensive evaluation of the analysed genes was performed in this study. Specifically, a statistically significant ( p  < 0.05) higher burden of the analysed genes harbouring at least one rare and damaging variant was detected in EM patients in comparison to healthy individuals. This result, considering the polygenic nature of EM, could be an intriguing breakthrough into the entangled EM genetic architecture, highlighting the potentially relevant involvement of these genes in this disease’s aetiology.\n\nIn conclusion, this study relied on a comprehensive, deep clinical evaluation and a WES analysis approach that, for the first time in the literature, allowed for the identification of novel and accurate genotype–phenotype correlations in an EM cohort, with a potential translational value into clinical practice. In-depth characterisation of the variants identified will be needed to confirm their biological relevance in EM onset and progression, thereby laying the foundation, in a long-term perspective, for the definition of novel and tailored treatment strategies for the better clinical management of EM patients.\nMoreover, three promising candidate genes (i.e.,  ABCA13 ,  NEB , and  CSMD1 ) were detected, allowing for new genetic insights into EM aetiopathogenesis. Replication studies in independent cohorts and functional experiments will be needed to further characterise the roles of these genes and variants to gain a deeper understanding of their effects in relation to EM.\nIn 30/80 (37.5%) of the analysed patients, no damaging variants within the selected genes were detected; this suggests that other players might be involved, underlying how the genetic landscape underlying EM is extremely complex.\nIn this light, the combined approach of an accurate clinical characterisation and a careful analysis of genetic data toward new candidates could be a successful strategy to identify novel molecular markers that, in the future, will make a substantial contribution to improving diagnostic and treatment strategies, thereby paving the way for personalised clinical management.","source_license":"CC0","license_restricted":false}