All-in-one exome sequencing approach for genetic testing of unexplained premature ovarian insufficiency

Is exome sequencing (ES) an efficient approach for simultaneous analysis of causative single gene defects and copy number variants (CNV) in unexplained premature ovarian insufficiency (POI)?

Among 51 idiopathic POI cases, a conservative molecular diagnostic yield of 12% was achieved with equal contribution of pathogenic or likely pathogenic (P/LP) monogenic causes and pathogenic microdeletions.

POI is a clinically and genetically heterogeneous condition, yet most cases remain idiopathic. Although numerous monogenic causes and CNVs have been implicated in POI, standardized clinical guidelines for comprehensive genetic analysis are still lacking.

This observational study of 51 unexplained POI cases implemented an all-in-one ES-based approach to analyze P and LP variants in 288 POI candidate genes and large (>0.5 Mb) pathogenic CNVs leading to POI.

Patients were recruited and phenotyped at two tertiary care women's health and endocrinology centers in Estonia. Bioinformatic processing of ES data and assessment of rare causal variants in POI were performed using an in-house analysis pipeline including automated filtering and manual assessment for pathogenicity, followed by experimental validation via Sanger sequencing (monogenic findings) and chromosomal microarray analysis (CNVs).

Six of 51 unexplained POI cases had confident molecular findings, including three cases with P/LP monogenic variants and three carrying large pathogenic CNVs. Overall molecular diagnostic yield was estimated to be 12% (6 of 51). There was a statistically significant overrepresentation of likely causal genetic findings in primary amenorrhea (5 of 9 cases, 56%) compared to secondary amenorrhea/oligomenorrhea (1 of 42 cases, 2%) (P = 3.0 × 10^-4). Primary amenorrhea cases presented diverse findings-TP63 p.(Arg97Gly) in three sisters (including one identified by cascade screening), 15q25.2 microdeletion in two unrelated subjects (POI relevant haploinsufficient genes: CPEB1, BNC1), and NR5A1 p.(Gly328Arg) in one case. A secondary amenorrhea case with ∼8.5 Mb microdeletion at Xq27.3-Xq28 enabled us to narrow down a critical region for non-recurrent Xq terminal losses causing POI (relevant haploinsufficient genes FMR1, AFF2, CETN2, HAUS7, and EMD). Additionally, heterozygous variants TBX6 c.622-2A>T, EXO1 c.136del, and NR2F2 p.(Val307Ala), as well as 1q21.1 microdeletion (two carriers), 1p36.22, and 12q21.1 microduplications were classified as potentially interesting Variants of Uncertain Significance (VUS) that require validation before their causal link to isolated POI can be assigned. TBX6 c.622-2A>T and 1q21.1 del have been implicated in other primary phenotypes, Mayer-Rokitansky-Küster-Hauser syndrome (MRKH, Müllerian anomalies), and 1q21.1 microdeletion syndrome (MIM #612474), respectively. A novel variant NR2F2 p.(Val307Ala) was identified in an isolated oligomenorrhea patient; the same substitution was independently detected in an unrelated male subject presenting oligozoospermia and unilateral cryptorchidism. No autosomal recessive POI cases were identified in our cohort, supporting a high level of heterogeneity and population-specificity in the genetic etiologies of POI, likely shaped by diverse demographic histories.

All variants linked identified in this study have been submitted to the NCBI ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar/) and FerGI (https://www.eshre.eu/Specialty-groups/Special-Interest-Groups/Reproductive-Genetics/FeRGI) databases.

All recruited participants were of white European ancestry and living in Estonia. Thus, the results might not apply to other ethnic groups. This study was conducted in a relatively small and well-selected cohort. Validation in larger and more diverse cohorts is needed to further assess the utility of ES in solving idiopathic POI cases.

The study's findings support the efficient use of ES as a comprehensive, all-in-one genetic test to achieve molecular diagnosis in unexplained POI, capturing both monogenic variants and pathogenic CNVs. Expanded genetic testing in POI is conceptually and clinically justified to reduce idiopathic cases and enable timely personalized management of reproductive and general health, including cascade testing of at-risk relatives. Given that ES enables the detection of both monogenic variants and CNVs, stepwise strategies may no longer be the most optimal, underscoring an urgent need to develop a standardized pipeline and guidelines for the generation, analysis, and interpretation of NGS data as well as for the counseling and management of patients based on their molecular findings.

This study was funded by the Estonian Research Council grants PRG1021 and PRG3041 (to M.L.).

The authors declare no conflicts of interest.