{'fraction_non_alphanumeric': 0.03771213073538655, 'fraction_numerical': 0.02885549397177304, 'mean_word_length': 5.094011142061281, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 6, 'https://': 1, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 1, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Novel Homozygous PADI6 Variants in Infertile Females with Early Embryonic Arrest

Early embryonic arrest denotes premature termination of development in preimplantation embryos, which is one of the major phenotypes of recurrent assisted reproduction failure. Padi6 is proven to be a member of the subcortical maternal complex (SCMC) in mice, which is essential in oocyte maturation and embryogenesis. We and other groups previously found that biallelic mutations in PADI6 caused female infertility manifesting as early embryonic arrest. In this study, we identified two novel homozygous variants (p.Cys163Arg, and p. Trp475*) of PADI6 in two infertile patients from a cohort of 75 females with the phenotype of early embryonic arrest. An in vitro expression study indicated severe decrease of PADI6, which might destruct the stability of SCMC. Our study expands the mutational spectrum of PADI6 and further supports the causality between PADI6 mutations and female infertility.


INTRODUCTION
The prevalence of infertility is estimated to be 8%-12% among reproductive-aged couples worldwide (Vander Borght and Wyns, 2018). The increasing number of infertile people accelerates the process of an aging population, which has become a serious social issue (Datta et al., 2016). With the help of assisted reproductive technology (ART), millions of couples with fertility problems deliver babies after 1-2 cycles of treatment. However, there is still a large number of people suffering from unexplained recurrent ART failure, among which early embryonic arrest accounts for a considerable proportion.
Early embryonic arrest is characterized by premature termination of development in preimplantation embryos. Its genetic basis was largely unknown for a long time. In the recent 5 years, with the advance of high-throughput sequencing technology, a group of pathogenic genes were identified, and biallelic mutations in PADI6 (610363), NLRP2 (609364), NLRP5 (609658), and FBXO43 (609110) were gradually reported to be responsible for the phenotype (Xu et al., 2016;Mu et al., 2019;Wang et al., 2021). PADI6, NLRP2, and NLRP5 are considered to form a subcortical maternal complex (SCMC), a multiprotein complex first identified in mice, which plays a critical role in essential events during the oocyte-to-embryo transition, including maternal RNA metabolism, mitochondrial rearrangement, and zygote genome activation (ZGA) (Lu et al., 2017). In 2016, we first reported biallelic mutations in PADI6 causing a lack of PADI6 in oocytes, which impaired the ZGA process of early embryonic development (Xu et al., 2016). Subsequently, studies from three independent groups strengthened the correlation between PADI6 variants and early embryonic arrest (Wang et al., 2018;Zheng et al., 2020;Liu et al., 2021). However, despite the previous findings, the pathogeny of the majority of patients was still unclear, and the genetic basis of human early embryonic development remained to be investigated.
Here, we recruited 75 Chinese patients diagnosed with early embryonic arrest in addition to the subjects studied in 2016 and identified two novel homozygous variants in PADI6 (p.Cys163Arg, and p. Trp475*). We investigated and compared the effects of the two variants with our previously identified variants (p.His211Gln, p. Gln324*, p. Gln381*, and p. Glu670Glyfs*48). Our study broadens the mutational spectrum of PADI6 and provides the further relationship between PADI6 variants and female infertility.

Genetic Analysis
Genomic DNA of all participants was extracted from peripheral blood using the HiPure Blood DNA Mini Kit (Magen Biotechnology, China) and was sheared to acquire 150-200 bp fragments by Biorupter (Diagenode, Belgium). The ends repair and adaptor ligation was performed using the Fast Library Prep Kit (iGeneTech, China). Whole exons were captured with the AIExome Enrichment Kit V1 (iGeneTech, China), and sequencing was performed on the Illumina platform. FastQC filtered clean reads were mapped to the reference genome (hg19) using Bwa. After removing duplications, SNV and InDel were called and annotated using GATK. For the patient from the consanguineous family, homozygosity mapping was performed to identify homozygous regions using HomozygosityMapper (Seelow et al., 2009). Variants were filtered according to the following criteria: 1) not recorded or recorded to have a frequency less than 0.1% in four public databases (000 Genomes Project [1,000 g], ExAC, gnomAD, and the NHLBI Exome Sequencing Project Exome Variant Server [ESP6500]); 2) missense, nonsense, splice-site, and indel variants; 3) homozygous variants in the identified homozygous regions; 4) predicted to be damaging by at least two in silico prediction models (SIFT, PROVEAN, PolyPhen-2); 5) genes were highly or specifically expressed in oocytes and early embryos based on our in-house database; 6) genes previously reported to affect female fertility in human or mouse. Candidate genes were validated by Sanger sequencing. The co-segregation validated genes were then screened in the sporadic cases in our cohort.

Plasmid Construction and Mutagenesis
The wild-type PADI6 coding sequence was cloned and inserted into the pRK7 vector with the N-terminal Flag tag. The mutant plasmids were generated using the Mut Express MultiS Fast Mutagenesis Kit (Vazyme Biotech Co.,Ltd, China). All the constructs were confirmed by Sanger sequencing.

Cell Culture and Transfection
Human embryonic kidney 293T (HEK-293T) cells were cultured in Dulbecco's Modified Eagle's Medium (Sigma-Aldrich) with 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco). Transient transfections were performed using the PolyJet DNA Transfection Reagent (SignaGen Laboratories, United States) according to the manufacturers' instructions. The pEGFP-C1 plasmid was co-transfected as controls. Three independent transfections were performed as the biological replicates.

RNA Isolation and Quantitative RT-PCR
Total RNA was isolated from 293T cells 36 h after transfection using TRIzol reagent, and 500 ng RNA was used for reverse transcription using the Evo M-MLV RT Mix Kit (Accurate Biotechnology, AG11728, China). Quantitative RT-PCR was performed using the SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology, AG11718, China) and QuantStudio 5 Real-Time PCR Systems (Thermo Fisher Scientific). All reactions were performed in at least biological triplicates with technical duplicates. The primers used in the experiments are listed in Supplementary Table S1. The relative expression level was calculated by 2 −ΔΔCt normalized by endogenous GAPDH expression.

Western Blot Analysis
Forty-eight hours after transfection, cells were harvested and lysed with RIPA lysis buffer containing 1% protease inhibitor cocktail (Bimake, United States). After 30 min incubating at 4°C, protein lysates were denatured in SDS protein loading buffer.

Identification of Novel Variants in PADI6
Whole-exome sequencing (WES), combined with homozygosity mapping, was first performed on the patient from the consanguineous family. Variant filtering was then conducted following our criteria. The first three criteria excluded most of the variants, and only 11 variants were retained in the gene list.  Figure 1A, left). The variant was located in a 3.2 Mb homozygous region (Chr1:17235196-20510690, Figure 1B, left), and was absent in ExAC, gnomAD, ESP6500, and 1000G database ( Table 1). It was predicted to be damaging or deleterious by SIFT, PROVEAN, and PolyPhen-2 ( Table 1). The mutant amino acid was located in the PAD middle domain of PADI6 and was highly conserved among different species (Figure 2A).
We subsequently screened exons of PADI6 in the cohort of 74 sporadic patients and identified one homozygous c.1425G > A [p.Trp475*] variant in PADI6 ( Figure 1A, right). Surprisingly, the patient denied that she was from a consanguineous family when we provided genetic counseling before she was included in our study. To confirm the causality and clarify the inheritance pattern of the variant, we first performed Sanger sequencing and confirmed the heterozygosity of her parents ( Figure 1A, right). Then, we performed WES and homozygosity mapping on the patient, and the result indicated that her parents might have a consanguineous marriage, and the variant was located in a 2 Mb homozygous region on chromosome 1 (Chr1:17517064-19452948, Figure 1B, right). None of the previously reported causative gene variants were identified in the WES data. The variant was absent in the four public databases and was predicted to be deleterious by PROVEAN ( Table 1). The Trp475 and the nearby residues were highly conserved among species (Figure 2A).

In Silico Prediction of the Variant Effects on Protein Function
The p. Trp475* variant was located in the functional proteinarginine deiminase (PAD) domain, and the p. Cys163Arg variant was located in the PAD middle domain of PADI6 (Figure 2A). These two variants were located in the highly conserved regions among different species (Figure 2A). The p. Cys163Arg variant was predicted to be damaging or deleterious by SIFT, PROVEAN, and PolyPhen-2, and the p. Trp475* variant was predicted to be deleterious by PROVEAN (Table 1). According to the AlphaFold predicted PADI6 structure, the wild-type Cys163 was located in the core region of the protein. Arginine substitution produced a prolonged side chain with a positive charge, which might disrupt the surrounding environment and result in a conformational change and misfolding of protein ( Figure 2B).

Clinical Phenotypes of the Patients
The patient from family 1 was from a consanguineous family. She was 33 years old and was diagnosed with primary infertility 6 years ago. She had experienced two failed ART attempts. In the first ART cycle, six oocytes were retrieved, and four matured oocytes were injected with sperm. Three oocytes were fertilized normally but were arrested at the zygote stage ( Figure 3A). In the second cycle, one MII oocyte was retrieved and was inseminated by ICSI. However, only one pro-nuclear (PN) was observed on day 1, and the egg was discarded ( Table 2). The patient from family 2 was 29 years old and had not conceived with regular unprotected sexual intercourse for 5 years. During the ART treatment, a total of 44 oocytes were retrieved, and 39 MII oocytes were inseminated by ICSI. Among them, 26 oocytes were fertilized and cleaved normally. However, only two usable embryos were acquired on day 3, and most of the embryos were arrested before the eight-cell stage ( Figure 3A). It is noteworthy that the   patient has a sister diagnosed with primary infertility for several years, and the infertile couple eventually adopted a child after the failure of several years of pregnancy attempt. We performed Sanger sequencing on the sister and identified the homozygous c.1425G > A variant in PADI6 ( Figure 1A, right, II-1).

PADI6 Variants Affected Protein Expression in HEK-293T Cells
We constructed PADI6 wild-type and mutant plasmids (two variants identified in this study and four variants identified in our previous study), and transiently transfected the constructs into HEK-293T cells to evaluate the functional influences of PADI6 variants in vitro. Immunoblot examination showed that, compared with wild-type PADI6, the expression level of p. Cys163Arg protein was significantly reduced. The p. Trp475* protein was observed at the expected smaller size with significantly reduced expression ( Figures 3B,C). The variants identified in our previous study displayed the same reduction in protein expression level, which was consistent with our previous findings in patients' oocytes ( Figures 3B,C) (Xu et al., 2016). Quantitative RT-PCR showed a significant reduction in mRNA levels in p. Trp475*, p. Gln324*, and p. Gln381* variants (Supplementary Figure S2).

DISCUSSION
In this study, we identified two novel homozygous variants (c.487T > C [p.Cys163Arg], and c.1425G > A [p.Trp475*]) in PADI6 in two infertile patients from a cohort of 75 females with the phenotype of early embryonic arrest, which caused recurrent ART failure. We transfected mutant constructs in HEK-293T cells to investigate the potential functional influences caused by the two variants. The result suggests that the two variants could cause significant reduction in protein expression in vitro, which is similar to the pathogenic variants identified in our previous study.
PADI6 is a member of the peptidylarginine deiminase (PADI) family, a family of calcium-dependent post-translational modify enzymes that can catalyze the process of citrullination by converting positively charged arginine residues to neutrally charged citrulline (Horibata et al., 2012). The PADI family includes five isozymes, PADI1, PADI2, PADI3, PADI4, and PADI6. Each PADI has biased expression among different tissues and has been proven to play critical roles in multiple diseases, including rheumatoid arthritis, Alzheimer's disease, ulcerative colitis, and cancers (Horibata et al., 2012;Mondal and Thompson, 2019;Curran et al., 2020;Al-U'datt et al., 2021). PADI6 is uniquely and highly expressed in different stages of oocytes and cleavage stage embryos, which suggests its specific role in oocyte maturation and early embryonic development (Xu et al., 2016).
During mammalian oocyte and early embryonic development, a group of maternal effect genes were found to play critical roles in regulating oocyte maturation, fertilization, and early embryonic development by modifying multiple processes, including maternal mRNA clearance, chromatin remodeling, and zygote genome activation (Kim and Lee, 2014). Among them, a multiprotein complex called SCMC was recently identified in mice and was proven to be essential for mouse cleavage-stage embryogenesis (Lu et al., 2017). The knockout mouse models of SCMC members were infertile and displayed embryonic developmental attest at the two-cell stage (Tong et al., 2000;Li et al., 2008;Yurttas et al., 2008;Yu et al., 2014). Padi6 was considered to be a member of SCMC, and eggs ovulated by female mice lack of Padi6 could be fertilized but could not develop beyond the cleavage stage (Wright et al., 2003;Esposito et al., 2007).
In our previous study, we found that the PADI6 mutations could lead to the reduction of PADI6 expression in patients' oocytes, which could impair the ZGA process and finally cause the developmental arrest of embryos (Xu et al., 2016). In this study, due to the limitation of clinical samples, we did not investigate the in vivo expression of PADI6 variants in patients' oocytes. Instead, we conducted the in vitro expression assay and compared the expression level of p. Cys163Arg and p. Trp475* variants with the wild-type and the pathogenic variants identified in our previous study: p. His211Gln, p. Gln324*, p. Gln381*, and p. Glu670Glyfs*48. The two variants displayed a significant reduction in protein expression, which was similar to the previously identified variants. Additionally, the mRNA levels were significantly reduced in p. Trp475*, p. Gln324*, and p. Gln381* variants, suggesting that the premature termination codons might cause the degradation of mRNAs by nonsensemediated mRNA decay (NMD), thus resulting in significantly reduced truncated proteins eventually. Because the p. Cys163Arg variant was a missense variant, we performed a 3-D structural prediction to provide supporting evidence of the pathogenicity of the variant. The wild-type Cys163 was located in the central core area of protein, and replacement of the noncharged cysteine residue by positive-charged arginine could alter the charge balance of the local area, which might lead to misfolding and degradation of the protein. We speculated that the variants could cause a significant reduction of PADI6 in oocytes, which might destroy the SCMC structural and functional stability, further affect the ZGA process of the early embryos, and finally lead to developmental arrest. Therefore, an effective supplement of PADI6 protein or mRNA in oocytes might rescue the phenotype, which is highly promising to investigate knockout mice in further study.
To summarize, we identified two novel homozygous variants (c.487T > C [p.Cys163Arg], and c.1425G > A [p.Trp475*]) in PADI6 in two females experiencing recurrent embryo developmental arrest. Our study expands the spectrum of genetic defects in female early embryonic arrest, and PADI6 is worth being screened in females with similar phenotypes.

DATA AVAILABILITY STATEMENT
The data sets presented in this study can be found in online repositories. The name of the repository and accession number can be found below: China National GeneBank DataBase (CNGBdb), https://db.cngb.org/, CNP0002647.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the Ethical committee of Shanghai East Hospital and Shanghai First Maternity and Infant Hospital. The patients/ participants provided their written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS
YX, LJ, and SX conceived and designed this study. YX, LS, and HY recruited the patients. RW, YL, YZ, and SX contributed to embryo evaluation and imaging. YX, RW, and ZP collected the clinical samples and clinical data. YX analyzed the sequencing data and performed the experiments. YX, ZW, GW, SL, LJ, and SX drafted and revised the manuscript. All the authors contributed to the article and approved the submitted version.