Targeted Next-Generation Sequencing Indicates a Frequent Oligogenic Involvement in Primary Ovarian Insufficiency Onset

Abstract

Primary ovarian insufficiency (POI) is one of the major causes of female infertility associated with the premature loss of ovarian function in about 3.7% of women before the age of 40. This disorder is highly heterogeneous and can manifest with a wide range of clinical phenotypes, ranging from ovarian dysgenesis and primary amenorrhea to post-pubertal secondary amenorrhea, with elevated serum gonadotropins and hypoestrogenism. The ovarian defect still remains idiopathic in some cases; however, a strong genetic component has been demonstrated by the next-generation sequencing (NGS) approach of familiar and sporadic POI cases. As recent evidence suggested an oligogenic architecture for POI, we developed a target NGS panel with 295 genes including known candidates and novel genetic determinants potentially involved in POI pathogenesis. Sixty-four patients with early onset POI (range: 10-25 years) of our cohort have been screened with 90% of target coverage at 50×. Here, we report 48 analyzed patients with at least one genetic variant (75%) in the selected candidate genes. In particular, we found the following: 11/64 patients (17%) with two variants, 9/64 (14%) with three variants, 9/64 (14%) with four variants, 3/64 (5%) with five variants, and 2/64 (3%) with six variants. The most severe phenotypes were associated with either the major number of variations or a worse prediction in pathogenicity of variants. Bioinformatic gene ontology analysis identified the following major pathways likely affected by gene variants: 1) cell cycle, meiosis, and DNA repair; 2) extracellular matrix remodeling; 3) reproduction; 4) cell metabolism; 5) cell proliferation; 6) calcium homeostasis; 7) NOTCH signaling; 8) signal transduction; 9) WNT signaling; 10) cell death; and 11) ubiquitin modifications. Consistently, the identified pathways have been described in other studies dissecting the mechanisms of folliculogenesis in animal models of altered fertility. In conclusion, our results contribute to define POI as an oligogenic disease and suggest novel candidates to be investigated in patients with POI.

Keywords: next-generation sequencing; oligogenic disease; primary amenorrhea; primary ovarian insufficiency; secondary amenorrhea.

Figure 1

Graph representations of the number of variants identified by NGS analysis in two subgroups of primary ovarian insufficiency (POI) patients. For each patient, indicated are the ID number (x-axis) and the number of variants/genes found altered (1 = 1 variant/gene; 2 = 2 variants/gene). Each color represents a different pathway: cell cycle, meiosis, and DNA repair (orange); ECM remodeling (blue); reproduction (green); cell metabolism (cyan); cell proliferation (purple); calcium release (pink); NOTCH signaling (red); signal transduction (yellow); WNT signaling (light orange); cell death (gray); and ubiquitination (dark blue). (A) The histogram reports the variants found by screening 295 candidate genes included in the OVO-Array panel. (B) NGS results of the analysis for diagnostic purposes of nine known POI genes.

Figure 2

Percentages of the number of patients per number of variants. (A) Seventy-five percent of patients analyzed through the OVO-Array panel harbor one or more variants, left. Histogram representing the percentages of patients carrying variants, right. (B) Twenty-five percent of patients analyzed in only nine known POI genes.

Figure 3

Bubble chart of the pathways identified by the gene ontology analysis. The sample numerosity (n) is reported for each phenotype (46,XX OD, light red; PA, light blue; eSA, light yellow; and SA <25 years, light green). Pathways are represented by colored bubbles (refer to the legend on the right). The size of the bubbles depends on the number of variants identified in each pathway for each phenotype. The percentages within the bubbles indicate the ratio between the number of variants per pathway associated to a phenotype and the total of variants associated to that phenotype. 46,XX OD = ovarian dysgenesis with normal karyotype (46,XX); PA = primary amenorrhea with functional ovarian defect, eSA = secondary amenorrhea onset few months after menarche; SA = secondary amenorrhea <25 years of age.