© American College of Medical Genetics and Genomics ARTICLE A high-resolution X chromosome copy-number variation map in fertile females and women with primary ovarian insufficiency Svetlana A. Yatsenko, MD 1,2,3,4, Michelle Wood-Trageser, PhD1, Tianjiao Chu, PhD3, Huaiyang Jiang, MD3 and Aleksandar Rajkovic, MD, PhD1,2,3,4,5,6 Purpose: Sex-biased expression of genes on the X chromosome is sequences among POI females, highlighting structural differences accomplished by a complex mechanism of dosage regulation that between X chromosomes of fertile and POI females. Furthermore, leads to anatomical and physiological differences between males we discovered a ~4% carrier incidence for X-linked disorders and females. Copy-number variations (CNVs) may impact the among fertile women. human genome by either affecting gene dosage or disturbing a Conclusion: We constructed a high-resolution map of female- chromosome structural and/or functional integrity. specific CNVs that provides critical insights into the spectrum of Methods: We performed a high-resolution CNV profiling to human genetic variation, sex-specific disease risk factors, and investigate the X chromosome integrity in cohorts of 269 fertile reproductive potential. We discovered novel CNVs associated with females and 111 women affected with primary ovarian insufficiency ovarian dysfunction and support polygenic models for POI. (POI) and assessed CNVs impact into functional and nonfunctional genomic elements. Genetics in Medicine (2019) 21:2275–2284; https://doi.org/10.1038/s41436- Results: In POI patients, we observed a 2.5-fold enrichment for 019-0505-2 rare CNVs comprising ovary-expressed genes, and genes implicated in autoimmune response and apoptotic signaling. Moreover, there Keywords: X chromosome; primary ovarian insufficiency; high- was a higher prevalence of deletions encompassing genes that resolution copy-number map; X-linked disease; fertility escape X inactivation, noncoding RNAs, and intergenic DNA INTRODUCTION ceases prior to age 40. The X chromosome aberrations are Sex chromosomes play a critical role in human fertility and encountered in 8–10% of individuals with POI and include sex determination. The X chromosome is enriched for sex- monosomy X, cytogenetically visible deletions and duplica- biased genes implicated in human reproduction, and tions, and balanced and unbalanced X-autosome rearrange- pathogenic variants within TEX11 (MIM *300311), ADGRG2 ments.7 Monosomy X (45,X or Turner syndrome) is (MIM *300572), AR (MIM *313700), and other X-linked characterized by loss of oocytes at the pachytene stage of – genes are known to impact male fertility.1 3 The X meiotic prophase during female fetal development, resulting chromosome is also critical in ovarian development and its in streak gonads at birth.8 The presence of two structurally disruption can lead to primary ovarian insufficiency (POI), normal X chromosomes is essential for meiosis, and meiotic which is clinically characterized by menstrual disturbance disturbance can lead to accelerated germ cell depletion, (amenorrhea or oligomenorrhea) with elevated follicle- apoptosis of surrounding somatic granulosa cells, and in the stimulating hormone(FSH) and luteinizing hormone and most severe form causes streak ovaries.5,8,9 Although one of – low estradiol (hypergonadotropic hypogonadism).4 7 The POI the X chromosomes is inactive in somatic cells, both X – spectrum is broad, affects 2–4% of women,4 6 and ranges chromosomes are active in ovaries and biallelic expression is – from congenital absence of ovaries (gonadal dysgenesis), likely important for the completion of meiosis.8 11 Moreover, which clinically presents as primary amenorrhea, to indivi- structural abnormalities, such as X chromosome/autosome duals with irregular menstrual cycles whose ovarian function translocations, and large X chromosome deletions on both the 1Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA; 2Department of Obstetrics,Gynecology and Reproductive Sciences, University of Pittsburgh, Pittsburgh, PA, USA; 3Magee-Womens Research Institute, Pittsburgh, PA, USA; 4Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA, USA; 5Department of Pathology, University of California–San Francisco, San Francisco, CA, USA; 6Department of Obstetrics, Gynecology and Reproductive Sciences, University of California San Francisco, San Francisco, CA, USA. Correspondence: Svetlana A. Yatsenko ([email protected]) or Aleksandar Rajkovic ([email protected]) Submitted 5 December 2018; accepted: 20 March 2019 Published online: 5 April 2019 GENETICS in MEDICINE | Volume 21 | Number 10 | October 2019 2275 ARTICLE YATSENKO et al short and long arms, have been associated with POI and performed two-tailed Wilcoxon rank sum tests. The values of infertility.12,13 In the majority of POI patients with X the statistical tests are further adjusted using the Holm chromosome translocations, the breakpoints do not contain method to control the family-wise error rates for the – genes.12 14 Despite clinical studies that associate large tracts of simultaneous test of all types of CNVs. X chromosome with POI, only few genes on the X chromosome have been implicated in ovarian development, RNA-Seq library preparation including BMP15 (MIM *300247), FMR1 (MIM *309550), The ovary RNA-Seq library was constructed using a 21-week- and PGRMC1 (MIM *300435). These observations suggest old human fetal ovary. The study was approved by the that failure in both the maintenance of the X chromosome University of Pittsburgh IRB (PRO16070068). – dosage and integrity can cause POI.12 14 The X chromosome copy-number profile in fertile women and women with POI is RESULTS unknown. We conducted a high-resolution analysis of copy- X chromosome CNVs in a population of fertile women number variants (CNVs) on 269 fertile women and 111 A total of 4099 CNVs (Table S1) comprising 2523 losses/ women with POI to establish a fine-scale X chromosome deletions (62%) and 1576 gains/duplications (38%) were architecture in these two populations, to evaluate their impact detected in 601 distinct genomic intervals, meaning that a on the X chromosome integrity, and to assess CNVs number of CNVs recurred (Table S2). CNVs analysis showed association with POI and female reproductive success. that 86% of the DNA losses were less than 10 kb in size (Fig. 1a). MATERIALS AND METHODS We classified copy-number alterations into six different The recruitment of participants and research study were categories (Fig. 1b). In the nonrepetitive genomic regions, the approved by the University of Pittsburgh institutional CNVs were classified as 411 homozygous losses (DNA copy 1234567890():,; review board (IRB) (PRO09080427, PRO12120172). Con- number = 0), 2074 heterozygous deletions (copy number = 1), sent for genetic testing was received from all patients. A 914 heterozygous duplications (copy number = 3), and 641 population of fertile females (269 unrelated participants) of CNVs with gains of two or more DNA copies (copy number European descent aged 23 to 37, who had at least one live ≥4). In regions with segmental duplications and/or gene family birth, have regular menses and no family history of genetic clusters, we recorded 38 partial losses and 21 partial gains. disorders, was defined as healthy women. One hundred and A total of 601 genomic intervals were defined by unique eleven POI women were defined as individuals diagnosed breakpoints observed among recorded CNVs. About 89.1% with hypergonadotropic hypogonadism, less than 40 years (3652/4099) of CNVs recurred with a population frequency of of age, and two serum FSH concentrations greater than greater than 1%. A total of 447/4099 (10.9%) CNVs were rare. 40 IU/L. The POI cohort had four subgroups: 34 women These 447 CNVs were detected in 189/269 (70.3%) of fertile with primary amenorrhea, 21 women with secondary women with an average of 2.36 (447/189) CNVs per amenorrhea diagnosed prior to age 25, 30 women with individual, and an overall incidence of 1.66 (447/269) rare secondary amenorrhea diagnosed after age 25, and CNVs per fertile female. Four or more rare CNVs were 26 women with POI, whose age of onset was prior to 40 observed in ~12% of fertile females (Fig. 1c). The majority but exact onset was uncertain. High-resolution (~0.5–3kb) (87%) of rare CNVs were heterozygous, with ~60% survey15 of the X chromosome CNVs was performed using representing DNA losses (mean size 13.9 kb) and ~40% a custom 180 K comparative genomic hybridization (CGH) containing DNA gains (mean size 130.7 kb). microarray (Agilent Inc., Santa Clara, CA) on genomic We examined all deletions and duplications intervals DNA from 269 fertile women and 111 POI study for the presence of genes and known regulatory elements. participants. The CNV calls are provided in Supplementary Whole genes or exons were present in 166/601 (~27.6%) Tables S1 and S7. Additional technical information including genomic intervals. In fertile females, the majority of detailed description of study subjects, microarray design, the gene-coding intervals are gains (67.4%) as opposed to bioinformatics and genomic tools, and associated references losses (32.6%). The majority of “loss” intervals (~81%) span are available in the Supplementary Material and Methods. intergenic and intronic regions, locations that are less Raw data are available in the National Center for Biotechnol- likely to be harmful. Only 30.5% of fertile females contain ogy