The human knockout phenotype of <i><scp>PADI6</scp></i> is female sterility caused by cleavage failure of their fertilized eggs

Naturally occurring human knockouts have greatly expanded our understanding of the medical relevance of genes, or lack thereof 1. Indeed, while biallelic inactivation of genes can result in severe Mendelian disorders, other genes seem to be tolerated when knocked out 2. We have been studying embryonic lethality as an extreme Mendelian phenotypic consequence of human knockout events by taking advantage of the consanguineous nature of the Saudi population where autozygosity facilitates the homozygous occurrence of alleles, including inactivating ones. We have previously shown an example of very early embryonic lethality caused by failure of homozygously TLE6 inactivated eggs to undergo the first cleavage after fertilization 3. In this communication, we report the identification of a very similar phenotype caused by PADI6 homozygous inactivation. The index is a 34-year-old Saudi lady who presented with her third cousin husband with primary infertility. Two intracytoplasmic sperm injection (ICSI) cycles produced a total of seven fertilized eggs successfully. However, none of these zygotes underwent the first cleavage except for one embryo in which fragmentation could not be ruled out as a cause of the apparent division. The couple was deemed eligible to our IRB-approved research protocol (KFSHRC RAC# 2121053) on the genetic causes of very early embryonic lethality and were recruited with informed consent. Blood was obtained from the index and available siblings, followed by DNA extraction, single nucleotide polymorphism (SNP) genotyping for autozygosity mapping and whole-exome sequencing as described before 4. By only considering novel (using our in-house database of 2379 Saudi exomes, and ExAC) homozygous coding/splicing variants within runs of autozygosity, we identified a single surviving variant: PADI6:NM_207421.3:c.1369C>T:p.(Arg457*) (NC_000001.10:g.17721477C>T). Segregation of this variant with the disease in the family was confirmed using Sanger sequencing (Fig. 1). PADI6 encodes a member of the peptidylarginine deiminase family that is highly enriched in the oocyte 5. Very similarly to Tle6−/−, Padi6−/− mice display female-specific sterility caused by an arrest of their embryos at the two-cell stage 6. The mechanism for this developmental arrest is thought to be related to disruption of zygotic gene activation as indicated by the reduced levels of its faithful readout phosphorylated RNA polymerase II 6. The link we propose between the very early embryonic lethality we observe in the embryos of our patient and her biallelic inactivation of PADI6 based on the strikingly similar mouse phenotype is further supported by the recent finding of Xu et al. 5. In their study of infertile couples with early cleavage arrest, Xu et al. 5 identified several biallelic inactivating mutations in PADI6 and showed that the arrested embryos have reduced levels of phosphorylated RNA polymerase II. Their finding that some embryos had their arrest at the two-, four- and even five-cell stage, which is later than observed in our patient and in the knockout mouse, may represent variability in the phenotype. Taken together, our findings and those by Xu et al. strongly indicate that PADI6 is yet another gene loss of which can cause very early embryonic lethality in humans, and that PADI6 and TLE6 should be screened in infertile couples with this phenotype. We thank the family for their enthusiastic participation. We thank the Genotyping and Sequencing Core Facilities at KFSHRC for their technical help. This work was supported by KACST grant 13-BIO1113-20 (FSA). S. Maddirevulaa S. Coskunb K. Awartanic H. Alsaifa F.M. Abdulwahaba F.S. Alkurayaa,d aDepartment of Genetics, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia bDepartment of Pathology and Laboratory Medicine, King Faisal Specialist Hospital and Research Center and College of Medicine, Alfaisal University, Riyadh, Saudi Arabia cDepartment of Obstetrics and Gynecology King Faisal Specialist Hospital and Research Center Riyadh, Saudi Arabia dDepartment of Anatomy, Cell Biology College of Medicine, Alfaisal University Riyadh, Saudi Arabia