Open Access

Whole exome sequencing identifies hemizygous deletions in the UGT2B28 and USP17L2 genes in a three‑generation family with endometriosis

  • Authors:
    • Hans M. Albertsen
    • Charoula Matalliotaki
    • Michail Matalliotakis
    • Maria I. Zervou
    • Ioannis Matalliotakis
    • Demetrios A. Spandidos
    • Rakesh Chettier
    • Kenneth Ward
    • George N. Goulielmos
  • View Affiliations

  • Published online on: January 3, 2019     https://doi.org/10.3892/mmr.2019.9818
  • Pages: 1716-1720
  • Copyright: © Albertsen et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

Endometriosis is an enigmatic condition with an unknown etiology and a poorly understood pathogenesis. It is considered to appear from the interplay of many genetic and environmental factors, affecting up to 10% of women and represents a major cause of pain and infertility. The familial association of endometriosis, as demonstrated through monozygotic twin and family studies suggests a genetic contribution to the disease, with further case‑control and genome‑wide association studies (GWAS) detecting various endometriosis risk factors. In a recent study, we described a unique, three‑generation family of Cretan origin (Greece) with 7 females with surgically confirmed endometriosis (grandmother, 3 daughters and 3 granddaughters). All the affected members of this family displayed a variety of clinical manifestations and complications. In the present study, to further analyze the genetic variants conferring the risk of developing endometriosis, whole exome sequencing (WES) was performed, using the AmpliSeq technology on the Ion Proton platform. An initial analysis of 64 variants that were detected across the 14 genes previously confirmed to be associated with endometriosis, did not identify any deleterious exonic variants in these genes. However, further analysis revealed 2 hemizygous deletions in the grandmother that segregate in several of her affected offspring. The first deletion was found in the UGT2B28 locus, spanning 7 informative sequence variants across at least 14 kb. The second deletion, located in USP17L2, spans 3 informative variants across at least 2 kb. On the whole, the findings of the presents study implicate 2 additional genes in the pathogenesis of endometriosis, apart from those already identified by GWAS.

Introduction

Endometriosis is a benign, common gynecologic disorder, affecting 6–10% of women during their reproductive years (1). Chronic pelvic pain, dyspareunia, dysmenorrhea and infertility represent the main manifestations (2). The absence of a timely and non-invasive diagnostic tool is presently the greatest barrier to the identification and treatment of endometriosis.

Endometriosis is characterized by a multifactorial pattern of inheritance, influenced by multiple genetic and environmental factors, as documented initially by the elevated incidence of severe endometriosis in the first-degree relatives of affected women compared to women lacking a positive family history (3,4). Furthermore, two monozygotic twin-based and family studies have demonstrated an approximately 50% heritability for endometriosis (5,6). Case-control studies, genome-wide association studies (GWAS) and meta-analyses have led to the identification of disease-risk loci that alter a woman's risk of developing the disorder and provide new insight into potential pathways leading to endometriosis. Particularly, GWAS in several series of samples have identified single nucleotide polymorphisms (SNPs) that are either directly or indirectly implicated in matrix remodeling, cell cycle regulation and signaling, cell adhesion, hormone receptors and metabolism, transcription regulation and inflammation, as well as immune and oxidative stress processes (7). However, all these data only account for approximately 5% of the disease variance (8). It is possible that rare and recent familial mutations, not detectable by GWAS, are responsible for part of the missing heritability. Thus, recent findings have suggested an increased burden of rare coding variants in genes involved in the development of complex diseases, apart from the established common variants identified by GWAS (9). Despite the continued progress being made in resolving the genetic underpinnings of endometriosis, a comprehensive list of genetic loci is far from being completed, and the functional role of causal variants remain poorly understood.

Recent progress in next generation sequencing provides us with the opportunity to search for less common variants with significant effects. In line with the aforementioned assumptions, whole exome sequencing (WES) provides a hypothesis-neutral approach. Accordingly, WES is a procedure that allows for the purification by sequence capture of all exonic regions of a genome and their further processing by next-generation sequencing (NGS) (10). Thus, prompted by the data collected by an initial genetic analysis (11) of a three-generation family of 7 affected women with surgically confirmed endometriosis and aiming to explore the contribution of functional coding variants to this disease, in the present study, we used WES to identify inherited rare, endometriosis-associated exonic variants in the patients of this family.

Patients and methods

Patients and study design

A three-generation family with 7 affected members is shown in Fig. 1. Case no. 1 was the first individual in the family to be diagnosed with endometriosis and underwent surgical hysterectomy at the age of 32 due to stage IV bilateral ovarian endometriosis. Her mother (not shown in the pedigree) had 4 children with no gynecological problems. However, her 3 daughters (case nos. 2–4) and 3 granddaughters (case nos. 5–7) have all been surgically diagnosed with endometriosis (11,12). Further clinical details regarding the disease severity of the affected members of the family have been previously reported in the study by Matalliotakis et al (11). In addition to endometriosis, case no. 1 has been diagnosed with 14 other morbidities, including Crohn's disease, interstitial cystitis, bronchial asthma, cardiovascular diseases, lupus erythematosus and multiple sclerosis (12).

The Ethics Committee of Venizeleio General Hospital of Heraklion (Heraklion, Greece) (ECHR no. 46/6686) approved the overall study and written informed consent was obtained from all the patients. The medical records were collected by the clinicians and pathologists, including surgical procedures and findings.

WES

Venous blood samples (5 ml per patient) were collected from the family members and stored at −20°C. Genomic DNA was isolated from peripheral blood leukocytes by using a commercial kit (PureLink® Genomic DNA mini kit; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions. Exome sequencing was conducted using the AmpliSeq technology on Ion Proton platform (Thermo Fisher Scientific, Inc.). A series of filters was applied in order to reduce sequencing artifacts. Following sequence assembly using Torrent software, variant annotation was performed using ANNOVAR (hg19 reference; http://annovar.openbioinformatics.org/en/latest/). Variants were determined using Ion Proton protocol and confirmed using the Genome Analysis Toolkit pipeline. Identity-by-descent (IBD) was calculated using PLINK (1.07) (13) and compared to the expected values for all relative-pairs in the pedigree.

Results

Common endometriosis-associated genetic variants

In the framework of a preliminary analysis conducted (data not shown), we first detected 64 variants across the 14 genes confirmed thus far to be associated with endometriosis (8). However, the vast majority of these variants were common in the population [minor allele frequency (MAF) >1%)] and, as a consequence, are not considered to be disease-causing. Moreover, we did not find any evidence of any deletions in these well-analyzed 14 genes.

Two rare variants (rs763439987 and rs139078629) were observed in the FN1 gene (MAF <1%), with each of the variants present in a single member of the family. Segregation analysis revealed that both variants appeared to be paternally inherited and in linkage disequilibrium (LD) (detailed data not shown).

Novel genetic variants

In a further in-depth analysis of normally segregating rare variants, we identified approximately 20,000 exonic variants in each of the 7 individuals, and almost 34,000 variants combined across the pedigree, which was in line with our expectations. IBD and segregation analysis confirmed all individual associations and the overall pedigree structure. We identified 2 hemizygous deletions segregating in this three-generation family (Fig. 2). A deletion was found in UDP glucuronosyltransferase family 2 member B28 (UGT2B28) genomic region, spanning 7 informative sequence variants across at least 14 kb, as well as a deletion in ubiquitin specific peptidase 17-like family member 2 (USP17L2) genomic region spanning 3 informative variants across at least 2 kb. Both deletions were present in the affected grandmother and segregated in as many as 4 and 5 of her descendants, respectively.

Discussion

Recent advances in genome sequencing technologies provide many opportunities for the characterization of individual genomic landscapes and for the identification of mutations relevant for diagnosis and therapy. In particular, WES has become a popular approach in the human genetics community considering its moderate costs, the amount of data that can be managed reasonably and the straightforward interpretation of the accumulated results upon analysis. The progress of high-throughput sequencing has led geneticists to directly perform WES for identifying the candidate variants in monogenic, as well as multifactorial diseases (14), for elucidating the genetic basis of some genetically heterogeneous disorders (15) and for an improved diagnosis of patients (16). In this framework, we hypothesized that WES of the affected members of a rare, three-generation family with 7 members with endometriosis, would be more likely to facilitate the identification of common and rare deleterious genetic variants that contribute to disease risk.

The current analysis demonstrated the absence of any deleterious exonic variants in genes confirmed thus far to be associated with endometriosis (8). However, the results obtained implicate the UGT2B28 and USP17L2 genes in the pathogenesis of endometriosis. Neither of the genes has previously been reported to be associated with endometriosis, at least to the best of our knowledge. UGT2B28 is a member of the UGT2B gene family, which is known to be involved in the metabolism of steroid hormones and several other xenobiotics (17) and is widely expressed in the liver, as well as extrahepatic steroid target tissues (18). Germ line copy-number variation in the UGT2B28 gene has been associated with Addison's disease (19), while common polymorphisms and copy number variation of the gene affect steroid hormone abundance and has been implicated in the increase of prostate cancer risk (18,20). Deletions of this gene have been reported to be predictors of the prostate-specific antigen (PSA) level in clinically localized prostate cancer following surgical treatment (21). Based on the aforementioned findings, the important role of UGT2B28 in the regulation of the hormonal exposure of steroid target cells has been suggested, and it has also been suggested that the alteration of UGT2B28 function may affect the metabolism of sex hormones (22). However, the mechanisms through which UGT2B28 may affect the pathogenesis of endometriosis are not yet clear.

USP17L2 (also known as DUB3, DUB-3 or USP17) is a member of the USP17 subfamily of USPs, consisted by cysteine proteases (23) regulating key cellular processes, such as cell growth and survival (24). It is transiently induced in response to cytokines and, when it is constitutively expressed, can block growth factor-dependent proliferation (24). USP17L2 has deubiquitinating activity and its overexpression leads to apoptosis and cell death (25). Ubiquitin has been found to be expressed in endometriotic cells and, as suggested, may contribute to a reduced sensitivity of ectopic endometrial tissue to apoptosis (26). Thus far, USP17L2 has been suggested to possibly be a valuable biomarker for the prediction of ovarian cancer prognosis and its inhibition may be a potential strategy for ovarian cancer treatment (27). This gene plays a central role in the regulation of the transcription factors, Slug and Twist. Particularly, it has been found that USP17L2 interacts with and stabilizes Slug and Twist through de-ubiquitination (28). As is known, Slug, Snail and Twist are three key epithelial-to-mesenchymal transition transcription factors (EMT-TFs) that are tightly regulated via ubiquitination and degradation. Furthermore, the aberrant expression/activation of EMT-TFs can give rise to tumor angiogenesis, invasion and metastasis (29,30).

EMT can be considered as a cellular de-differentiation process providing cells with the increased plasticity required during embryonic development and tissue remodeling, due to the similarities in the migratory and invasive characteristics that the cells obtain during these processes (31,32). We have previously proposed that the pathogenesis of endometriosis involves the loss of mesothelial barrier integrity due to the activation of the EMT repair mechanism (33). As a consequence, in the absence of the mesothelial barrier, endometrial cells can more readily adhere to the underlying peritoneal stroma and establish endometrial lesions. Thus, we speculate that the dosage-dependent loss of USP17L2 affects mesothelial integrity and increases the risk of developing endometriosis.

Furthermore, in the framework of the present WES, two rare variants (rs763439987 and rs139078629) were observed in the FN1 gene to be paternally inherited. Although both of the hemizygous deletions clearly are inherited from the grandmother of the family under investigation, any paternally inherited risk-variants, such as the rare FN1 haplotype, must also be considered.

In conclusion, the present study identified, for the first time, to the best of our knowledge, a role of the UGT2B28 and USP17L2 genes in endometriosis and underlines the power of WES to decipher complex phenotypes. However, the precise underlying mechanisms remain elusive. The present findings may enhance our biological insight of endometriosis and may contribute towards novel strategies regarding improved patient care. Further in-depth analysis of normally segregating rare variants is ongoing.

Acknowledgements

Not applicable.

Funding

Juneau Biosciences provided funding for the sequencing.

Availability of data and materials

All data generated or analyzed during the present study are included in this published article or are available from the corresponding author on reasonable request.

Authors' contributions

HMA, RC, KW, GNG and CM conceived and designed the study and drafted the manuscript. HMA, RC, KW and MIZ performed the experiments. MM, RC, CM, GNG and MIZ searched the literature. IM, CM and MM obtained the clinical data. CM, RC, MIZ, DAS, GNG and IM analyzed and interpreted the data. DAS, HMA, MM, MIZ and IM critically revised the manuscript. All authors have read and approved the final manuscript.

Ethics approval and consent to participate

The Ethics Committees of the Human Research at Venizeleio General Hospital of Heraklion (ECHR no. 46/6686) approved the overall study and written informed consent was obtained from all the patients.

Patient consent for publication

Not applicable.

Competing interests

HMA, RC and KW are employed by Juneau Biosciences who provided salary support for the study. DAS is the Editor-in-Chief for the journal, but had no personal involvement in the reviewing process, or any influence in terms of adjudicating on the final decision, for this article.

References

1 

Burney RO and Giudice LC: Pathogenesis and pathophysiology of endometriosis. Fertil Steril. 98:511–519. 2012. View Article : Google Scholar : PubMed/NCBI

2 

Halis G and Arici A: Endometriosis and inflammation in infertility. Ann N Y Acad Sci 1034. 300–315. 2004. View Article : Google Scholar

3 

Simpson JL, Elias S, Malinak LR and Buttram VC Jr: Heritable aspects of endometriosis. I. Genetic studies. Am J Obstet Gynecol. 137:327–331. 1980. View Article : Google Scholar : PubMed/NCBI

4 

Hadfield RM, Mardon HJ, Barlow DH and Kennedy SH: Endometriosis in monozygotic twins. Fertil Steril. 68:941–942. 1997. View Article : Google Scholar : PubMed/NCBI

5 

Treloar SA, O'Connor DT, O'Connor VM and Martin NG: Genetic influences on endometriosis in an Australian twin sample. simplesueT@qimr.edu.au. Fertil Steril. 71:701–710. 1999. View Article : Google Scholar : PubMed/NCBI

6 

Saha R, Pettersson HJ, Svedberg P, Olovsson M, Bergqvist A, Marions L, Tornvall P and Kuja-Halkola R: Heritability of endometriosis. Fertil Steril. 104:947–952. 2015. View Article : Google Scholar : PubMed/NCBI

7 

Kobayashi H, Imanaka S, Nakamura H and Tsuji A: Understanding the role of epigenomic, genomic and genetic alterations in the development of endometriosis (review). Mol Med Rep. 9:1483–1505. 2014. View Article : Google Scholar : PubMed/NCBI

8 

Sapkota Y, Steinthorsdottir V, Morris AP, Fassbender A, Rahmioglu N, De Vivo I, Buring JE, Zhang F, Edwards TL, Jones S, et al iPSYCH-SSI-Broad Group, : Meta-analysis identifies five novel loci associated with endometriosis highlighting key genes involved in hormone metabolism. Nat Commun. 8:155392017. View Article : Google Scholar : PubMed/NCBI

9 

Gao L, Emond MJ, Louie T, Cheadle C, Berger AE, Rafaels N, Vergara C, Kim Y, Taub MA, Ruczinski I, et al National Heart, Lung, and Blood Institute GO Exome Sequencing Project, : Identification of rare variants in ATP8B4 as a risk factor for systemic sclerosis by whole-exome sequencing. Arthritis Rheumatol. 68:191–200. 2016. View Article : Google Scholar : PubMed/NCBI

10 

Ng SB, Turner EH, Robertson PD, Flygare SD, Bigham AW, Lee C, Shaffer T, Wong M, Bhattacharjee A, Eichler EE, et al: Targeted capture and massively parallel sequencing of 12 human exomes. Nature. 461:272–276. 2009. View Article : Google Scholar : PubMed/NCBI

11 

Matalliotakis M, Zervou MI, Matalliotaki C, Arici A, Spandidos DA, Matalliotakis I and Goulielmos GN: Genetic association study in a three-generation family with seven members with endometriosis. Mol Med Rep. 16:6077–6080. 2017. View Article : Google Scholar : PubMed/NCBI

12 

Matalliotaki C, Matalliotakis M, Zervou MI, Trivli A, Matalliotakis I, Mavromatidis G, Spandidos DA, Albertsen HM, Chettier R, Ward K and Goulielmos GN: Co-existence of endometriosis with 13 non-gynecological co-morbidities: Mutation analysis by whole exome sequencing. Mol Med Rep. 18:5053–5057. 2018.PubMed/NCBI

13 

Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D, Maller J, Sklar P, de Bakker PI, Daly MJ and Sham PC: PLINK: A tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet. 81:559–575. 2007. View Article : Google Scholar : PubMed/NCBI

14 

Ng SB, Buckingham KJ, Lee C, Bigham AW, Tabor HK, Dent KM, Huff CD, Shannon PT, Jabs EW, Nickerson DA, et al: Exome sequencing identifies the cause of a mendelian disorder. Nat Genet. 42:30–35. 2010. View Article : Google Scholar : PubMed/NCBI

15 

Girard SL, Gauthier J, Noreau A, Xiong L, Zhou S, Jouan L, Dionne-Laporte A, Spiegelman D, Henrion E, Diallo O, et al: Increased exonic de novo mutation rate in individuals with schizophrenia. Nat Genet. 43:860–863. 2011. View Article : Google Scholar : PubMed/NCBI

16 

Choi M, Scholl UI, Ji W, Liu T, Tikhonova IR, Zumbo P, Nayir A, Bakkaloğlu A, Ozen S, Sanjad S, et al: Genetic diagnosis by whole exome capture and massively parallel DNA sequencing. Proc Natl Acad Sci USA. 106:19096–19101. 2009. View Article : Google Scholar : PubMed/NCBI

17 

Tephly TR and Burchell B: UDP-glucuronosyltransferases: A family of detoxifying enzymes. Trends Pharmacol Sci. 11:276–279. 1990. View Article : Google Scholar : PubMed/NCBI

18 

Guillemette C, Lévesque E, Harvey M, Bellemare J and Menard V: UGT genomic diversity: Beyond gene duplication. Drug Metab Rev. 42:24–44. 2010. View Article : Google Scholar : PubMed/NCBI

19 

Brønstad I, Wolff AS, Løvås K, Knappskog PM and Husebye ES: Genome-wide copy number variation (CNV) in patients with autoimmune Addison's disease. BMC Med Genet. 12:1112011. View Article : Google Scholar : PubMed/NCBI

20 

Karypidis AH, Olsson M, Andersson S-O, Rane A and Ekström L: Deletion polymorphism of the UGT2B17 gene is associated with increased risk for prostate cancer and correlated to gene expression in the prostate. Pharmacogenomics J. 8:147–151. 2008. View Article : Google Scholar : PubMed/NCBI

21 

Nadeau G, Bellemare J, Audet-Walsh É, Flageole C, Huang SP, Bao BY, Douville P, Caron P, Fradet Y, Lacombe L, et al: Deletions of the androgen-metabolizing UGT2B genes have an effect on circulating steroid levels and biochemical recurrence after radical prostatectomy in localized prostate cancer. J Clin Endocrinol Metab. 96:E1550–E1557. 2011. View Article : Google Scholar : PubMed/NCBI

22 

Belledant A, Hovington H, Garcia L, Caron P, Brisson H, Villeneuve L, Simonyan D, Têtu B, Fradet Y, Lacombe L, et al: The UGT2B28 sex-steroid inactivation pathway is a regulator of steroidogenesis and modifies the risk of prostate cancer progression. Eur Urol. 69:601–609. 2016. View Article : Google Scholar : PubMed/NCBI

23 

Nijman SM, Luna-Vargas MP, Velds A, Brummelkamp TR, Dirac AM, Sixma TK and Bernards R: A genomic and functional inventory of deubiquitinating enzymes. Cell. 123:773–786. 2005. View Article : Google Scholar : PubMed/NCBI

24 

Burrows JF, McGrattan MJ, Rascle A, Humbert M, Baek KH and Johnston JA: DUB-3, a cytokine-inducible deubiquitinating enzyme that blocks proliferation. J Biol Chem. 279:13993–14000. 2004. View Article : Google Scholar : PubMed/NCBI

25 

Shin JM, Yoo KJ, Kim MS, Kim D and Baek KH: Hyaluronan- and RNA-binding deubiquitinating enzymes of USP17 family members associated with cell viability. BMC Genomics. 7:2922006. View Article : Google Scholar : PubMed/NCBI

26 

Ilad RS, Fleming SD, Bebington CR and Murphy CR: Ubiquitin is associated with the survival of ectopic stromal cells in endometriosis. Reprod Biol Endocrinol. 2:692004. View Article : Google Scholar : PubMed/NCBI

27 

Zhou B, Shu B, Xi T, Su N and Liu J: Dub3 expression correlates with tumor progression and poor prognosis in human epithelial ovarian cancer. Biomed Pharmacother. 70:84–89. 2015. View Article : Google Scholar : PubMed/NCBI

28 

Lin Y, Wang Y, Shi Q, Yu Q, Liu C, Feng J, Deng J, Evers BM, Zhou BP and Wu Y: Stabilization of the transcription factors slug and twist by the deubiquitinase dub3 is a key requirement for tumor metastasis. Oncotarget. 8:75127–75140. 2017.PubMed/NCBI

29 

Blanco MJ, Moreno-Bueno G, Sarrio D, Locascio A, Cano A, Palacios J and Nieto MA: Correlation of Snail expression with histological grade and lymph node status in breast carcinomas. Oncogene. 21:3241–3246. 2002. View Article : Google Scholar : PubMed/NCBI

30 

Zhou BP, Deng J, Xia W, Xu J, Li YM, Gunduz M and Hung MC: Dual regulation of Snail by GSK-3beta-mediated phosphorylation in control of epithelial-mesenchymal transition. Nat Cell Biol. 6:931–940. 2004. View Article : Google Scholar : PubMed/NCBI

31 

Thiery JP and Sleeman JP: Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol. 7:131–142. 2006. View Article : Google Scholar : PubMed/NCBI

32 

Thiery JP, Acloque H, Huang RY and Nieto MA: Epithelial-mesenchymal transitions in development and disease. Cell. 139:871–890. 2009. View Article : Google Scholar : PubMed/NCBI

33 

Albertsen HM and Ward K: Genes linked to endometriosis by GWAS are integral to cytoskeleton regulation and suggests that mesothelial barrier homeostasis is a factor in the pathogenesis of endometriosis. Reprod Sci. 24:803–811. 2017. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

March 2019
Volume 19 Issue 3

Print ISSN: 1791-2997
Online ISSN:1791-3004

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
APA
Albertsen, H.M., Matalliotaki, C., Matalliotakis, M., Zervou, M.I., Matalliotakis, I., Spandidos, D.A. ... Goulielmos, G.N. (2019). Whole exome sequencing identifies hemizygous deletions in the UGT2B28 and USP17L2 genes in a three‑generation family with endometriosis. Molecular Medicine Reports, 19, 1716-1720. https://doi.org/10.3892/mmr.2019.9818
MLA
Albertsen, H. M., Matalliotaki, C., Matalliotakis, M., Zervou, M. I., Matalliotakis, I., Spandidos, D. A., Chettier, R., Ward, K., Goulielmos, G. N."Whole exome sequencing identifies hemizygous deletions in the UGT2B28 and USP17L2 genes in a three‑generation family with endometriosis". Molecular Medicine Reports 19.3 (2019): 1716-1720.
Chicago
Albertsen, H. M., Matalliotaki, C., Matalliotakis, M., Zervou, M. I., Matalliotakis, I., Spandidos, D. A., Chettier, R., Ward, K., Goulielmos, G. N."Whole exome sequencing identifies hemizygous deletions in the UGT2B28 and USP17L2 genes in a three‑generation family with endometriosis". Molecular Medicine Reports 19, no. 3 (2019): 1716-1720. https://doi.org/10.3892/mmr.2019.9818