Population-based genetic analysis in infertile men reveals novel mutations of <i>ADAD</i> family members in patients with impaired spermatogenesis

Dai, Siyu; Liu, Mohan; Liu, Man; Jiang, Chuan; Yang, Yanting; Han, Hong; Yang, Yihong; Jiang, Xiaohui; Shen, Ying

doi:10.1093/hmg/ddad012

Cited by 9 publications

(4 citation statements)

References 21 publications

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“…Our analyses show that loss of ADAD1 leads to reduced sperm motility along with distinct sperm head morphological defects, in particular reduced DNA compaction and overall head volume. A recent study suggests a similar impact of ADAD1 mutation in humans as individuals with pathogenic ADAD1 mutations present with reduced sperm motility and sperm head shape abnormalities [71]. Although Adad1 mutant testes show minimal gene expression changes, the dramatic reduction of ribosome association in numerous spermatid transcripts demonstrates that ADAD1 influences translation of specific transcripts.…”

Section: Discussionmentioning

confidence: 99%

ADAD1 is required for normal translation of nuclear pore and transport protein transcripts in spermatids of Mus musculus

Potgieter

Eddy

Badrinath

et al. 2023

Biology of Reproduction

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ADAD1 is a testis-specific RNA binding protein expressed in post-meiotic spermatids whose loss leads to defective sperm morphology and male infertility. However, the drivers of the Adad1 phenotype remain unclear. Morphological and functional analysis of Adad1 mutant sperm demonstrated defective DNA compaction, abnormal head shaping, and reduced motility. RNA sequencing of Adad1 mutant testes revealed minimal transcriptome changes however ribosome association of many transcripts was reduced, suggesting ADAD1 may be required for their translational activation. Additionally, immunofluorescence of proteins encoded by select transcripts showed delayed protein accumulation. Further analyses demonstrated impaired subcellular localization of multiple proteins suggesting protein transport is also abnormal in Adad1 mutants. To clarify the mechanism giving rise to this, the manchette, a protein transport microtubule network, and the LINC complex, which connects the manchette to the nuclear lamin, were assessed across spermatid development. Proteins of both displayed delayed translation and/or localization in mutant spermatids implicating ADAD1 in their regulation, even in the absence of altered ribosome association. Finally, ADAD1’s impact on the nuclear pore complex (NPC), a regulator of both the manchette and the LINC complex, was examined. Reduced ribosome association of NPC encoding transcripts and reduced NPC protein abundance along with abnormal localization in Adad1 mutants confirmed ADAD1 is required for normal translation of the NPC in post-meiotic germ cells. Together, these studies lead to a model whereby ADAD1’s influence on nuclear transport leads to deregulation of the LINC complex and the manchette, ultimately generating the range of physiological defects observed in the Adad1 phenotype. Summary sentence: ADAD1 is a post-meiotic spermatid RNA binding protein that is required for normal translation of mRNAs important for post-meiotic differentiation and mRNAs associated with nuclear and intracellular transport.

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Section: Discussionmentioning

confidence: 99%

ADAD1 is required for normal translation of nuclear pore and transport protein transcripts in spermatids of Mus musculus

Potgieter

Eddy

Badrinath

et al. 2023

Biology of Reproduction

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“…sqADAR/D-like does not appear to be cephalopod-specific as BLAST searches uncover similarities to the recently described ADAD-like molecules in other mollusks ( Zhang et al, 2023 ). ADADs act as RNA-binding molecules found to be essential for spermatogenesis in mammals ( Schumacher et al, 1995 ; Snyder et al, 2020 ; Dai et al, 2023 ). The function of ADAD-like molecules in mollusks is unknown.…”

Section: Discussionmentioning

confidence: 99%

Squid express conserved ADAR orthologs that possess novel features

et al. 2023

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The coleoid cephalopods display unusually extensive mRNA recoding by adenosine deamination, yet the underlying mechanisms are not well understood. Because the adenosine deaminases that act on RNA (ADAR) enzymes catalyze this form of RNA editing, the structure and function of the cephalopod orthologs may provide clues. Recent genome sequencing projects have provided blueprints for the full complement of coleoid cephalopod ADARs. Previous results from our laboratory have shown that squid express an ADAR2 homolog, with two splice variants named sqADAR2a and sqADAR2b and that these messages are extensively edited. Based on octopus and squid genomes, transcriptomes, and cDNA cloning, we discovered that two additional ADAR homologs are expressed in coleoids. The first is orthologous to vertebrate ADAR1. Unlike other ADAR1s, however, it contains a novel N-terminal domain of 641 aa that is predicted to be disordered, contains 67 phosphorylation motifs, and has an amino acid composition that is unusually high in serines and basic amino acids. mRNAs encoding sqADAR1 are themselves extensively edited. A third ADAR-like enzyme, sqADAR/D-like, which is not orthologous to any of the vertebrate isoforms, is also present. Messages encoding sqADAR/D-like are not edited. Studies using recombinant sqADARs suggest that only sqADAR1 and sqADAR2 are active adenosine deaminases, both on perfect duplex dsRNA and on a squid potassium channel mRNA substrate known to be edited in vivo. sqADAR/D-like shows no activity on these substrates. Overall, these results reveal some unique features in sqADARs that may contribute to the high-level RNA recoding observed in cephalopods.

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“…In Adad2 Mut/Mut mice, we observed very few elongated spermatids or spermatozoa, suggesting that the frameshift or missense variants of ADAD2 , located in the adenosine deaminase domain, are associated with a good prognosis for testicular sperm extraction, and that the retrieved sperm might be recovered and used for ICSI. However, a recent report described a patient with asthenoteratozoospermia carrying a homozygous missense mutation of ADAD2 (c.1381C>T, p.R461W), located in the adenosine deaminase domain, the same domain as our MT1 (MT1: c.G829T, p.G277C) and MT2 (c.G1192A, p.D398N), who failed to cause pregnancy even after ICSI treatment ( Dai et al , 2023 ), suggesting that the poor-quality spermatozoa from the ADAD2 mutant patient were not sufficient to father a biological child. Therefore, ROSI might be an alternative therapy for patients harboring ADAD2 mutations.…”

Section: Discussionmentioning

confidence: 99%

“…Previous studies have suggested that a homozygous stop-gain mutation in ADAD2 (c.1186C>T, p.Gln396Ter) and compound heterozygous mutations (Hg19: chr16:84012049–84224913del and c.82dupC, p.Gln28ProfsTer136) in ADAD2 might be associated with incomplete spermatogonial arrest ( Krausz et al , 2020 ). Patients with severe asthenoteratozoospermia carrying a homozygous frameshift mutation (c.17_18del, p.Gln6Argfs*3) or a homozygous missense mutation (c.1381C>T, p.Arg461Trp) in ADAD2 showed morphological deformities in both the sperm head and flagellum assembly ( Dai et al , 2023 ; Tian et al , 2023 ), suggesting that ADAD2 may affect human spermatogenesis. However, the reproductive phenotype of these ADAD2 mutations identified in humans differ from those in Adad2 ko mice, and the pathological effects of these ADAD2 mutations have not been verified in mouse models.…”

Section: Introductionmentioning

confidence: 99%

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

Shi,

Shah,

Liu

et al. 2023

Human Reproduction Open

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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 two 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 three 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 INTEREST(S) 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. WHAT DOES THIS MEAN FOR PATIENTS? Non-obstructive azoospermia (NOA) is characterized by impaired spermatogenesis and is the most severe form of male infertility. Approximately 25% of NOA cases are attributed to genetic anomalies, but only mutations in a small number of genes have been validated as pathogenic in NOA patients. Our study focused on six infertile patients with NOA who were carriers of three ADAD2 mutations from different and unrelated families. Functional evidence has shown that these mutations cause premature degradation of the ADAD2 protein and are associated with failure to of round spermatids to differentiate into spermatozoa during spermatogenesis. Despite the lack of spermatozoa in the testes, Adad2Mut/Mut mice with mutation efficiencies similar to that of human patients were able to produce healthy and fertile offspring after round spermatid injection. Therefore, our study provides a preliminary insight for the genetic counselling of couples with ADAD2-mutation-associated male infertility.

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Population-based genetic analysis in infertile men reveals novel mutations of ADAD family members in patients with impaired spermatogenesis

Cited by 9 publications

References 21 publications

ADAD1 is required for normal translation of nuclear pore and transport protein transcripts in spermatids of Mus musculus

ADAD1 is required for normal translation of nuclear pore and transport protein transcripts in spermatids of Mus musculus

Squid express conserved ADAR orthologs that possess novel features

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

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