Novel PNLDC1 mutations underlie nonobstructive azoospermia in humans and mice

PIWI-interacting RNAs (piRNAs) are small regulatory RNAs (21-35 nucleotides) exclusively expressed in germ cells, where they play a critical role in transposable element repression and post-meiotic gene regulation. The poly(A)-specific RNase-like domain-containing 1 (PNLDC1) protein is essential for piRNA maturation, specifically in 3'-end trimming. Disruption of PNLDC1 has been implicated in nonobstructive azoospermia (NOA) and male infertility. Through whole-exome sequencing, we identified a compound heterozygous mutation (MT1 c.449G > A, p.Trp150* and MT2 c.821A > G, p.His274Ala) in a Chinese NOA patient (P9241) and a homozygous nonsense mutation (MT3 c.1288C > T, p.Arg430*) in a Pakistani NOA patient (II:2) born to a consanguineous couple. Mutant PNLDC1 mRNA was detected, but not the corresponding protein, was detected in the testes of P9241. In contrast, truncated PNLDC1 protein was observed in HEK293T cells transfected with a plasmid harboring mutation MT3. To investigate the functional consequences, we generated a Pnldc1KI/KI mouse model mimicking the MT3 using CRISPR/Cas9 genome editing, which exhibited infertility due to spermiogenesis arrest, phenocopying the NOA condition in patient II:2. Notably, Pnldc1KI/KI testes showed significant derepression of the retrotransposon LINE1 and increased spermatid apoptosis. These findings provide strong functional evidence that PNLDC1 mutations disrupt piRNA biogenesis, impair spermatogenesis, and underlie NOA in both humans and mice.