Biallelic mutations in RNA-binding protein ADAD2 cause spermiogenic failure and non-obstructive azoospermia in humans.

IF 8.3 Q1 OBSTETRICS & GYNECOLOGY
Baolu Shi, Wasim Shah, Li Liu, Chenjia Gong, Jianteng Zhou, Tanveer Abbas, Hui Ma, Huan Zhang, Menglei Yang, Yuanwei Zhang, Nadeem Ullah, Zubair Mahammad, Mazhar Khan, Ghulam Murtaza, Asim Ali, Ranjha Khan, Jiahao Sha, Yan Yuan, Qinghua Shi
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In mice, the lack of the RNA-binding protein ADAD2 leads to a complete absence of sperm in epididymides due to failure of spemiogenesis, but the spermatogenic effects of <i>ADAD2</i> mutations in human NOA-associated infertility require functional verification.</p><p><strong>Study design size duration: </strong>Six infertile male patients from three unrelated families were diagnosed with NOA at local hospitals in Pakistan based on infertility history, sex hormone levels, two semen analyses and scrotal ultrasound. Testicular biopsies were performed in two of the six patients. <i>Adad2</i> mutant mice (<i>Adad2<sup>Mut/Mut</sup></i>) carrying mutations similar to those found in NOA patients were generated using the CRISPR/Cas9 genome editing tool. Reproductive phenotypes of <i>Adad2<sup>Mut/Mut</sup></i> mice were verified at 2 months of age. Round spermatids from the littermates of wild-type (WT) and <i>Adad2<sup>Mut/Mut</sup></i> mice were randomly selected and injected into stimulated WT oocytes. This round spermatid injection (ROSI) procedure was conducted with three biological replicates and >400 ROSI-derived zygotes were evaluated. The fertility of the ROSI-derived progeny was evaluated for three months in four <i>Adad2<sup>WT/Mut</sup></i> male mice and six <i>Adad2<sup>WT/Mut</sup></i> female mice. A total of 120 <i>Adad2<sup>Mut/Mut</sup></i>, <i>Adad2<sup>WT/Mut</sup></i>, and WT mice were used in this study. The entire study was conducted over 3 years.</p><p><strong>Participants/materials setting methods: </strong>Whole-exome sequencing was performed to detect potentially pathogenic mutations in the six NOA-affected patients. 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引用次数: 0

Abstract

Study question: What are some pathogenic mutations for non-obstructive azoospermia (NOA) and their effects on spermatogenesis?

Summary answer: Biallelic missense and frameshift mutations in ADAD2 disrupt the differentiation of round spermatids to spermatozoa causing azoospermia in humans and mice.

What is known already: NOA is the most severe cause of male infertility characterized by an absence of sperm in the ejaculate due to impairment of spermatogenesis. In mice, the lack of the RNA-binding protein ADAD2 leads to a complete absence of sperm in epididymides due to failure of spemiogenesis, but the spermatogenic effects of ADAD2 mutations in human NOA-associated infertility require functional verification.

Study design size duration: Six infertile male patients from three unrelated families were diagnosed with NOA at local hospitals in Pakistan based on infertility history, sex hormone levels, two semen analyses and scrotal ultrasound. Testicular biopsies were performed in two of the six patients. Adad2 mutant mice (Adad2Mut/Mut) carrying mutations similar to those found in NOA patients were generated using the CRISPR/Cas9 genome editing tool. Reproductive phenotypes of Adad2Mut/Mut mice were verified at 2 months of age. Round spermatids from the littermates of wild-type (WT) and Adad2Mut/Mut mice were randomly selected and injected into stimulated WT oocytes. This round spermatid injection (ROSI) procedure was conducted with three biological replicates and >400 ROSI-derived zygotes were evaluated. The fertility of the ROSI-derived progeny was evaluated for three months in four Adad2WT/Mut male mice and six Adad2WT/Mut female mice. A total of 120 Adad2Mut/Mut, Adad2WT/Mut, and WT mice were used in this study. The entire study was conducted over 3 years.

Participants/materials setting methods: Whole-exome sequencing was performed to detect potentially pathogenic mutations in the six NOA-affected patients. The pathogenicity of the identified ADAD2 mutations was assessed and validated in human testicular tissues and in mouse models recapitulating the mutations in the NOA patients using quantitative PCR, western blotting, hematoxylin-eosin staining, Periodic acid-Schiff staining, and immunofluorescence. Round spermatids of WT and Adad2Mut/Mut mice were collected by fluorescence-activated cell sorting and injected into stimulated WT oocytes. The development of ROSI-derived offspring was evaluated in the embryonic and postnatal stages.

Main results and the role of chance: Three recessive mutations were identified in ADAD2 (MT1: c.G829T, p.G277C; MT2: c.G1192A, p.D398N; MT3: c.917_918del, p.Q306Rfs*43) in patients from three unrelated Pakistani families. MT1 and MT2 dramatically reduced the testicular expression of ADAD2, likely causing spermiogenesis failure in the NOA patients. Immunofluorescence analysis of the Adad2Mut/Mut male mice with the corresponding MT3 mutation showed instability and premature degradation of the ADAD2 protein, resulting in the spermiogenesis deficiency phenotype. Through ROSI, the Adad2Mut/Mut mice could produce pups with comparable embryonic development (46.7% in Adad2Mut/Mut versus 50% in WT) and birth rates (21.45 ± 10.43% in Adad2Mut/Mut versus 27.5 ± 3.536% in WT, P = 0.5044) to WT mice. The Adad2WT/Mut progeny from ROSI (17 pups in total via three ROSI replicates) did not show overt developmental defects and had normal fertility.

Large scale data: N/A.

Limitations reasons for caution: This is a preliminary report suggesting that ROSI can be an effective treatment for infertile Adad2Mut/Mut mice. Further assisted reproductive attempts need to be carefully examined in humans during clinical trials.

Wider implications of the findings: Our work provides functional evidence that mutations in the ADAD2 gene are deleterious and cause consistent spermiogenic defects in both humans and mice. In addition, preliminary results show that ROSI can help Adad2Mut/Mut to produce biological progeny. These findings provide valuable clues for genetic counselling on the ADAD2 mutants-associated infertility in human males.

Study funding/competing interests: This work was supported by the National Natural Science Foundation of China (32000587, U21A20204, and 32061143006), and the National Key Research and Developmental Program of China (2019YFA0802600 and 2021YFC2700202). This work was also supported by Institute of Health and Medicine, Hefei Comprehensive National Science Center, Hefei, China. The authors declare no competing interests.

Abstract Image

Abstract Image

Abstract Image

rna结合蛋白ADAD2的双等位基因突变导致人类生精失败和非阻塞性无精子症。
研究问题:非阻塞性无精子症(NOA)有哪些致病突变及其对精子发生的影响?摘要:ADAD2的双等位基因错义和移码突变破坏了圆形精子向精子的分化,导致人类和小鼠无精子症。已知情况:NOA是男性不育的最严重原因,其特征是由于精子发生障碍而导致射精中没有精子。在小鼠中,由于精子发生失败,缺乏rna结合蛋白ADAD2导致附睾中完全没有精子,但ADAD2突变在人类noa相关不孕症中的生精作用需要功能验证。研究设计规模持续时间:根据不孕症史、性激素水平、两次精液分析和阴囊超声,来自三个无血缘关系家庭的6名不育男性患者在巴基斯坦当地医院被诊断为NOA。6例患者中2例行睾丸活检。使用CRISPR/Cas9基因组编辑工具生成了携带与NOA患者相似突变的Adad2突变小鼠(Adad2Mut/Mut)。在2月龄时验证Adad2Mut/Mut小鼠的生殖表型。从野生型(WT)和Adad2Mut/Mut小鼠的窝仔中随机抽取圆形精子,注射到受刺激的WT卵母细胞中。这种圆形精子注射(ROSI)过程进行了3次生物重复,并评估了超过400个ROSI衍生的受精卵。在4只Adad2WT/Mut雄性小鼠和6只Adad2WT/Mut雌性小鼠中,对rosi衍生后代的生育能力进行了为期3个月的评估。本研究共使用Adad2Mut/Mut、Adad2WT/Mut和WT小鼠120只。整个研究进行了三年多。参与者/材料设置方法:对6例noa患者进行全外显子组测序以检测潜在的致病性突变。通过定量PCR、western blotting、苏木精-伊红染色、周期性酸-希夫染色和免疫荧光等方法,对鉴定出的ADAD2突变在人睾丸组织和小鼠模型中的致病性进行了评估和验证。采用荧光活化细胞分选法收集WT和Adad2Mut/Mut小鼠的圆形精细胞,注入受刺激的WT卵母细胞。在胚胎和出生后阶段对rosi衍生后代的发育进行了评估。主要结果及其作用:在ADAD2中鉴定出3个隐性突变(MT1: c.G829T, p.G277C;MT2: c.G1192A, p.D398N;MT3: c.917_918del, p.Q306Rfs*43)。MT1和MT2显著降低了ADAD2的睾丸表达,可能导致NOA患者精子发生失败。免疫荧光分析相应MT3突变的Adad2Mut/Mut雄性小鼠显示ADAD2蛋白不稳定和过早降解,导致精子发生缺陷表型。通过ROSI, Adad2Mut/Mut小鼠产生的幼崽胚胎发育(Adad2Mut/Mut组为46.7%,WT组为50%)和出生率(Adad2Mut/Mut组为21.45±10.43%,WT组为27.5±3.536%,P = 0.5044)与WT小鼠相当。来自ROSI的Adad2WT/Mut后代(通过三个ROSI重复共17只幼崽)没有表现出明显的发育缺陷,并且具有正常的生育能力。大规模数据:无。局限性:这是一份初步报告,表明ROSI可以有效治疗不育的Adad2Mut/Mut小鼠。在临床试验期间,进一步的辅助生殖尝试需要在人类中仔细检查。研究结果的更广泛意义:我们的工作提供了功能证据,证明ADAD2基因的突变是有害的,并在人类和小鼠中引起一致的生精缺陷。此外,初步结果表明,ROSI可以帮助Adad2Mut/Mut产生生物后代。这些发现为人类男性ADAD2突变体相关不育的遗传咨询提供了有价值的线索。研究经费/利益竞争:国家自然科学基金项目(32000587,U21A20204, 32061143006)和国家重点研发计划项目(2019YFA0802600, 2021YFC2700202)资助。本研究也得到了中国合肥国家综合科学中心卫生与医学研究所的支持。作者声明没有利益冲突。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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