Functional and clinical insights into nuclear receptor variants for advancing precision diagnostics in male infertility.

Background: Nuclear receptors, including steroidogenic factor 1 (NR5A1/SF1) and the androgen receptor (AR), are transcription factors regulating physiological processes, e.g., reproduction. Pathogenic variants in these receptors are associated with a broad spectrum of phenotypes, ranging from differences in sexual development to isolated male infertility. However, standardised methods to classify variants of uncertain significance (VUS) in these genes are lacking, complicating diagnosis and individualised treatment.

Methods: We queried rare NR5A1 and AR variants in exome/genome sequencing data of 2127 infertile men. Pathogenicity assessment included thorough clinical phenotyping, familial segregation, in silico pathogenicity prediction combining traditional and machine-learning tools, and functional evaluation of the variants using in vitro assays.

Findings: We identified a total of seven heterozygous NR5A1 variants in 10 infertile men and 22 hemizygous AR variants in 31 infertile men with severe oligo-/azoospermia. Of these, three SF1 and seven AR variants displayed significantly reduced transcriptional activity. This study led to the reclassification of one NR5A1 variant and ten AR variants, including three AR variants that were reclassified from VUS to (likely) pathogenic. Combined phenotype, in silico, and in vitro data led to 60% of all variants (17 out of 29) being classified as (likely) pathogenic per ACMG guidelines, providing insights into the phenotypic features and spermatogenic impairment in affected men.

Interpretation: This study highlights the importance of combining clinical and experimental data for the assessment of VUS in nuclear receptors to reliably classify pathogenicity and to improve patient diagnosis and care.

Funding: This study was carried out within the frame of the Deutsche Forschungsgemeinschaft (DFG)-sponsored Clinical Research Unit 'Male Germ Cells' (CRU326, project 329621271, to F.T., C.F., S.L., and J.G.) and the German Federal Ministry of Education and Research (BMBF)-sponsored Junior Scientist Research Centre 'ReproTrack.MS' (grant 01GR2303, to F.T. and S.K.), and was supported by the Medical Faculty Münster via an Innovative Medical Research (IMF) grant (GA-122104, to A.S.G.) and the Clinician Scientist programme CareerS (to S.A.K.).