丹麦哥本哈根健康与医学学院Kristian Almstrup团队研究了变异型PNLDC1、piRNA加工缺陷和无精子症的相关性。该项研究成果发表在2021年8月4日出版的《新英格兰医学杂志》上。

P-元件诱导无用睾丸（PIWI）相互作用RNA（piRNAs）较短（长度为21至35个核苷酸）且不编码，几乎只存在于生殖细胞中，它们调节转座子的异常表达和减数分裂后的基因表达。对piRNAs的加工至关重要的是PNLDC1，该结构域修饰其3′端，当在小鼠中被破坏时，会导致无精子症和雄性不育。

研究组对924名被诊断为非梗阻性无精子症的男性的DNA样本进行了外显子组测序。睾丸活检标本通过组织学和免疫组化检测、原位杂交、逆转录酶定量聚合酶链反应和小RNA测序进行分析。

四名无血缘关系的中东裔男性患有非梗阻性无精子症，他们被发现携带PNLDC1突变：第一位患者有双等位基因停止-增益突变p.R452Ter；第二位有一种新的双等位错义突变p.P84S；第三位有p.M259T和p.L35PfsTer3组成的复合杂合突变；第四位有一种新的双等位基因典型剪接受体位点突变，c.607-2A→T。

睾丸组织学结果一致显示有错误减数分裂和生精阻滞，其中Sa型圆形精子细胞是最高级的生殖细胞群体。PNLDC1基因和蛋白表达以及piRNAs加工蛋白PIWIL1、PIWIL4、MYBL1和TDRKH在睾丸细胞中的表达明显减少。此外，在携带PNLDC1突变的男性中，piRNAs的长度分布和粗线期piRNAs的数量显著改变。

研究结果表明，错误的piRNA处理对男性减数分裂和精子发生有直接的机制作用，最终导致男性不育。

附：英文原文

Title: Variant PNLDC1, Defective piRNA Processing, and Azoospermia

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

Issue&Volume: 2021-08-04

Abstract:

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.

DOI: 10.1056/NEJMoa2028973

Source: https://www.nejm.org/doi/full/10.1056/NEJMoa2028973