Variant PNLDC1, Defective piRNA Processing, and Azoospermia

Liina Nagirnaja, Nina Mørup, John E Nielsen, Rytis Stakaitis, Ieva Golubickaite, Manon S Oud, Sofia B Winge, Filipa Carvalho, Kenneth I Aston, Francesca Khani, Godfried W van der Heijden, C Joana Marques, Niels E Skakkebaek, Ewa Rajpert-De Meyts, Peter N Schlegel, Niels Jørgensen, Joris A Veltman, Alexandra M Lopes, Donald F Conrad, Kristian Almstrup

25 Citations (Scopus)


BACKGROUND: P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are short (21 to 35 nucleotides in length) and noncoding and are found almost exclusively in germ cells, where they regulate aberrant expression of transposable elements and postmeiotic gene expression. Critical to the processing of piRNAs is the protein poly(A)-specific RNase-like domain containing 1 (PNLDC1), which trims their 3' ends and, when disrupted in mice, causes azoospermia and male infertility.

METHODS: We performed exome sequencing on DNA samples from 924 men who had received a diagnosis of nonobstructive azoospermia. Testicular-biopsy samples were analyzed by means of histologic and immunohistochemical tests, in situ hybridization, reverse-transcriptase-quantitative-polymerase-chain-reaction assay, and small-RNA sequencing.

RESULTS: Four unrelated men of Middle Eastern descent who had nonobstructive azoospermia were found to carry mutations in PNLDC1: the first patient had a biallelic stop-gain mutation, p.R452Ter (rs200629089; minor allele frequency, 0.00004); the second, a novel biallelic missense variant, p.P84S; the third, two compound heterozygous mutations consisting of p.M259T (rs141903829; minor allele frequency, 0.0007) and p.L35PfsTer3 (rs754159168; minor allele frequency, 0.00004); and the fourth, a novel biallelic canonical splice acceptor site variant, c.607-2A→T. Testicular histologic findings consistently showed error-prone meiosis and spermatogenic arrest with round spermatids of type Sa as the most advanced population of germ cells. Gene and protein expression of PNLDC1, as well as the piRNA-processing proteins PIWIL1, PIWIL4, MYBL1, and TDRKH, were greatly diminished in cells of the testes. Furthermore, the length distribution of piRNAs and the number of pachytene piRNAs was significantly altered in men carrying PNLDC1 mutations.

CONCLUSIONS: Our results suggest a direct mechanistic effect of faulty piRNA processing on meiosis and spermatogenesis in men, ultimately leading to male infertility. (Funded by Innovation Fund Denmark and others.).

Original languageEnglish
JournalThe New England journal of medicine
Issue number8
Pages (from-to)707-719
Number of pages13
Publication statusPublished - 19 Aug 2021


  • Adult
  • Azoospermia/genetics
  • Biopsy
  • Exoribonucleases/genetics
  • Gene Expression
  • Humans
  • Infertility, Male/genetics
  • Male
  • Meiosis/physiology
  • Mutation
  • Phenotype
  • Polymerase Chain Reaction
  • RNA, Small Interfering/metabolism
  • Sequence Analysis, RNA
  • Testis/metabolism
  • Whole Exome Sequencing


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