(743) RBBP7 Splicing and Missense Variants Leading to Non-Obstructive Azoospermia and Leydig Cell Tumors

Introduction Non-obstructive azoospermia (NOA), the most severe form of male infertility, is characterized by impaired spermatogenesis. Evidence suggests that X-linked genes play a pivotal role in this process, with the Retinoblastoma-Binding Protein 7 (RBBP7) gene being the most frequently implicated. RBBP7 encodes a histone H4-binding component of chromatin remodeling complexes. Loss-of-function variants in RBBP7 have been associated with spermatogenic maturation arrest and, in limited reports, with Leydig cell tumors; however, reproductive outcomes achieved via intracytoplasmic sperm injection (ICSI) remain scarcely documented. Objective To investigate the mechanistic roles of RBBP7 mutations (c.886-1G>T and c.962A>G) in male infertility and testicular tumorigenesis; to define their impact on protein function and spermatogenic maturation arrest; to assess the clinical efficacy of micro-TESE-ICSI intervention; and to evaluate RBBP7's potential as a diagnostic biomarker for NOA. Methods Whole-exome sequencing (WES) and Sanger sequencing were employed to identify pathogenic variants in infertile men. Pathogenicity and clinical significance of the identified variants were assessed through pedigree analysis, Minigene splicing assays, molecular predictive modeling, and correlation with micro-TESE/ICSI outcomes. Histological and immunohistochemical analyses were performed on micro-TESE-derived testicular tissues. Variant classification followed the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines. Results Both patients presented with persistent azoospermia. Patient 1 exhibited severely reduced testicular volume (1-2 ml) with hypoechoic areas on ultrasound. Following successful sperm retrieval via micro-TESE, a sustained clinical pregnancy was achieved through ICSI. Pathological examination revealed a Leydig-type stromal tumor. Patient 2 had testicular volumes of 2-3 ml and previous fertility history, but was lost to follow-up after diagnosis. Genetic analysis identified hemizygous RBBP7 mutations: a maternally inherited splice-site variant (c.886-1G>T) in Patient 1 and a missense mutation (c.962A>G, p.Q321R) in Patient 2. Functional characterization demonstrated that both variants disrupt normal splicing. The c.886-1G>T variant induced a 7-bp deletion in exon 8, causing a frameshift and premature termination codon, predicting a truncated 302-amino acid protein. The c.962A>G mutation resulted in complete exon 8 skipping, leading to an internal 26-amino acid deletion and a predicted 399-amino acid truncated protein. Conclusions This study confirms the association between RBBP7 mutations and NOA as well as Leydig cell tumors. It elucidates the molecular mechanisms by which the RBBP7 c.886-1G>T and c.962A>G p.Q321R mutations cause spermatogenic failure, likely through disrupted histone modification and DNA damage repair pathways. The role of mutant RBBP7 protein in testicular tumorigenesis warrants further exploration. These findings provide a basis for genetic counseling and clinical management strategies for patients with RBBP7-related NOA and Leydig-type stromal tumors. RBBP7 should be included in genetic testing panels for NOA. For patients with Disclosure No