Homozygous RRM2 Variant Might Lead to Early Embryo Developmental Arrest: A Case Report

Background: Early embryonic developmental stagnation is one of the reasons that affect the outcome of in vitro fertilization-embryo transfer, leading to the depletion of available embryos or failure after transplantation. It has been shown that defects in the ribonucleotide-reductase lead to cell cycle arrest, developmental delay, and high mutation rates. Case presentation: Two female patients, who were siblings from an inbreeding family, suffered primary infertility of unknown causes and early embryonic developmental arrest during IVF treatment. A total of 39 oocytes were obtained from the patients in collectively 5 IVF/ICSI cycles, of which 37 were mature eggs, only 2 transplantable embryos were formed, and no pregnancy was achieved. Whole genome sequencing and Sanger sequencing were adopted to identify and conrm variations that might cause early embryo developmental stagnation in this family. We identied a homozygous variant c.262C>T:p.His88Tyr in ribonucleotide-diphosphate reductase subunit M2 (RRM2) in both patients and their parents each carried a heterozygous allele. Pedigree analysis showed an autosomal recessive inheritance pattern. Function of this variant was predicted by online databases, which indicated it to be a potential pathogenic mutation. Conclusions: We identied RRM2 as a potential causative gene for early embryonic developmental stagnation. It was also suggested that RRM2 might be a maternal effect gene.


Background
Infertility is one serious issue that threatens the human reproductive health. According to statistics, on a worldwide scale 15% of married couples are infertile, and infertility factors affect about half of men and women (1). The development of assisted reproductive technology brings hope to many infertile patients. Studies have shown that 40%-70% of human embryos in in vitro fertilization (IVF) cycles are living embryos, whereas the others are arrested at different developmental stages (2). Stagnant embryos are usually discarded, and live embryos are frozen, continue to be cultured into blastocysts, or directly transplanted back into the patient's uterus. However, in some cases the development of all embryos from a patient are arrested at early developmental stages, leading to failed IVF attempt, which cannot be rescued by multiple embryo transplantation or single sperm microinjection. The cause of such condition is speculated to relate to genetic factors.
At present, research on the genetic factors of human embryonic stagnation is very limited. Up to now, there are only two reported gene mutations that could cause human embryo development stagnation and infertility. A homozygous variation in TLE6 gene was rst reported to account for the stagnation of early embryonic development in human, which was identi ed by exome sequencing in an Arab inbreeding family (3). TLE6 is a maternal effect gene. In the early stage of human embryo development, especially from fertilization to the 8-cell stage, gene transcription is not fully initiated. The basic biological processes of cells including protein synthesis and regulation of embryo development depend almost entirely on the RNA stored in the egg. Therefore, the mutation of maternal effect genes may affect the development and survival of fertilized eggs and embryos. In another study, homozygous mutations in PADI6 gene in a Chinese inbreeding family resulted in the stagnation of fertilized eggs and embryos from the female patient (4). In the meantime, compound heterozygous mutations of PADI6 gene were found in 2 of 36 sporadic cases of early embryonic development arrest (4). Interestingly, PADI6 is also a maternal effect gene, which again indicated the important role of maternal factors in early embryonic development.
In this study, we reported the identi cation of a homozygous variation NM_001034: exon3: c.262C > T: p.His88Tyr in ribonucleotide-diphosphate reductase subunit M2 (RRM2) in two infertile sisters from an inbreed family, who both suffered primary infertility and early embryo developmental arrest. RRM2 was also identi ed as a potential maternal effect factor.

Case Presentation
Patients Two female patients P1 and P2 (Table 1), who were siblings from an inbreeding family (Fig. 1A), were diagnosed of primary infertility. Both patients were in good physical condition, and had regular sexual intercourse of 2-3 times a week. Through physical examination, ultrasonography, hystero-salpingography and laparoscopic exploration, we found no abnormalities in the patients' vulva, vagina, cervix, ovary, fallopian tubes, and uterus. Periodic development of follicles was monitored by transvaginal ultrasound. Blood gonadal hormone was detected on the second day after menstruation, and no abnormalities were observed in all indicators in either patient. The patients and their husbands all had normal karyotype. The reproductive system of both patients' husband was well developed, and no abnormalities were found in semen analysis. The two couples had sought medical advice elsewhere but failed to identify any potential cause of infertility and had been treated with empirical Chinese herbal medicine. Both patients received in vitro fertilization and embryo transfer (IVF-ET) in our institution ( Table 2). Exome sequencing identi ed homozygous RRM2 variation We performed whole exome sequencing as previously described (3) on the peripheral blood sample of P1 and focused on the identi cation of homozygous variations. The average depth of whole exome sequencing was greater than 100×. Through screening the gene polymorphisms with allele frequencies greater than 1% in the dbSNP, 1000 Genome and ESP6500 databases, we found a list of genes containing homozygous variations (Table 3), among which only RRM2 is closely related to embryonic development potential. The homozygous variant in RRM2 (NM_001034:exon3:c.262C > T:p.His88Tyr) was con rmed by Sanger sequencing as previously described (5)   In silico analysis predicted that RRM2:c.262C > T was a disease-associated mutation in human ( Table 4). The allele frequency of RRM2:c.262C > T in the world population was very low, but was 10-fold higher in the East Asian population. However, homozygotes of this variant has not been documented in either 1000 Genomes or Exome Aggregation Consortium (ExAC) database. The c.262C > T point mutation in RRM2 resulted in a substitution of histidine by tyrosine at the 88 amino acid. As histidine is a hydrophilic, positively-charged basic amino acid while tyrosine is a hydrophobic, neutral amino acid with aromatic residue, this substitution might affect the three-dimensional protein conformation, attenuating the stability and function of RRM2 protein (Fig. 2B). This histidine of RRM2 is highly conserved among different species (Fig. 2C), indicating its functional importance. The heterozygous variant of RRM2:c.262C > T carried by the patients' parents suggested that heterozygote of this variant is not pathogenic. i Mutation Taster (http://www.mutationtaster.org/). The probability value is the probability of the prediction, that is, a value close to 1 indicates a high"security the prediction.

Discussion And Conclusions
This study identi ed the homozygous variant RRM2:c.262C > T:p.His88Tyr in two infertile patients that might be the potential cause of early embryonic development stagnation in their IVF-ET treatment, which resulted in depletion of all available embryos for transplantation. Bioinformatics analysis predicted this variant might affect RRM2 protein function and cause disease. According to the American College of Medical Genetics (ACMG) guidelines for classi cation of variants (6), this was classi ed as a "variant of uncertain signi cance".
Ribonucleotide reductase (RR) consists of two subunits (RRM1 and RRM2), and is a rate-limiting enzyme in deoxynucleotide production for DNA synthesis (7).
As optimal cell concentration of deoxyribonucleotides is essential for DNA synthesis, replication, and repair (8), RR plays an important role in cell proliferation. RR defects often lead to cell cycle arrest, developmental delay, and abnormally increased RR activities led to high frequency of gene mutation (9). Functionally, RRM1 controls substrate speci city, and RRM2 regulates the overall catalytic activity (10). RRM2 is widely expressed in human organs, with highest expression level in secondary oocyte (data from Bgee data Base for Gene Expression Evolution).
RRM2 could regulate zygotic genome activation (ZGA) through the yes-associated protein (YAP). In early mammalian embryos, genomic transcription is quiescent until ZGA occurs 2-3 days after fertilization. The coding gene of YAP, Yap1, was found to be highly expressed in human and mouse oocytes and early embryos (11,12), and maternally accumulated YAP in oocyte is essential for ZGA (1). The embryos of Yap1 knockout female mice presented prolonged 2-cell stage and slower development into the 4-cell stage. Rrm2, the mouse homologous gene of human RRM2, and Rpl13 were found to be the target genes of YAP in early blastomeres, which were required to mediate maternal YAP's effect in conferring developmental competence on preimplantation embryos (13). This was in accordance with our observation that the embryonic development stagnation caused by RRM2 variation exhibited maternal effect recessive inheritance. That is, female homozygotes for the RRM2 mutation appeared phenotypically normal, whereas their offspring shown the mutant phenotype of lethality during early embryonic development.
RRM2 has also been reported to play an important role in inducing cell proliferation and decidualization in mouse uterus, suggesting its likely involvement in early embryo development and embryo implantation. Embryo implantation into the maternal uterus is a crucial step in the successful pregnancy of mammals.
Currently, implantation and trophoblastic in ltration defects are major obstacles to successful pregnancy (14). Embryo implantation is a complex developmental process, including the process of embryo attachment, followed by invasion of the matrix, and proliferation and differentiation of endometrial stromal cells (15). It has been shown that Rrm2 is strongly expressed in decidual tissues and is up-regulated by progesterone and DNA damage in mouse (7).
RRM2-speci c inhibitors effectively reduced the weight of implantation sites and deciduoma (7). In the future, functional study by generating genetically modi ed mouse in the Rrm2 gene would help supporting the pathogenicity of this variant.
In summary, this study identi ed a homozygous variation of RRM2:exon3:c.262C > T:p.His88Tyr, which might alter RRM2 protein conformation and attenuate its function, and might be a potential cause of early embryonic development stagnation in two sisters who had multiple failed assisted reproduction attempts. This study also suggested that RRM2 might be a maternal effect gene. Further animal study is still required to con rm the role of RRM2 in early embryo development.