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Exome Sequencing Homes in on Gene Linked to Early Human Embryonic Lethality

NEW YORK (GenomeWeb) – By sequencing the exomes of women having difficulties conceiving, researchers in Saudi Arabia have discovered a gene that, when mutated, leads to the early death of embryos.

The team, led by King Faisal Specialist Hospital and Research Center's Fowzan Alkuraya, performed autozygosity mapping and exome sequencing on women from two consanguineous families who were have trouble conceiving even after in vitro fertilization efforts. As they reported in Genome Biology this week, the researchers found that a mutation in TLE6 appeared to stop embryonic development early on. Other genes active in later embryonic development have been linked to embryonic lethality, but this, the researchers said, is the first one identified that is active prior to implantation.

"Our data suggest that TLE6 mutations are a rare cause of human female-limited infertility and represent the earliest known human embryonic lethality that is explicable by a single gene mutation," Alkuraya and his colleagues wrote in their paper.

Failure of fertilization even with intracytoplasmic injection of apparently healthy sperm into apparently healthy eggs is rare, the researchers said, noting that in 20 years of IVF experience, they could only recall that occurring with eight couples. Two of those couples were consanguineous, and the researchers were able to re-contact them. One woman also had an affected sister, the researchers noted.

Both sisters from that family, which also included other brothers and sisters who were healthy and fertile, underwent multiple rounds of intra-cytoplasmic sperm injection. Only three oocytes developed two pronuclei, indicating normal fertilization, but these zygotes stopped developing at the one-, two-, and four-cell stages.

The woman from the other family exhibited a similar pattern, the researchers said, which indicates that the phenotype in these women was pre-implantation embryonic lethality.

For two of the three women, Alkuraya and his colleagues performed whole-exome sequencing to search for homozygous coding or splice variants within their autozygomes.

After these filters, one novel variant became apparent: a homozygous S510Y substitution in TLE6.

Further, the researchers reported that all three women shared this autozygous interval. As they all had an identical haplotype, this suggested they shared a common ancestor.

They also noted that a brother from one family was homozygous for this variant, though he was fertile, suggesting that the variant's effect is restricted to women.

The researchers reported that the mutated residue appears to be universally conserved among mammalian TLE6 orthologs, adding that both PolyPhen and SIFT predict the S510Y variant to be pathogenic.

TLE6 encodes a protein, Alkuraya and his colleagues noted, that is part of the sub-cortical maternal complex (SCMC), a structure in mammalian oocytes that is key to very early embryonic development. The gene, they added, is also one of the few known mammalian maternal effect genes.

Protein kinase A is known to phosphorylate TLE6, and the researchers suspected that the replacement of a serine residue could affect a phosphorylation site on TLE6.

Through a series of cell line and immunoblot analyses, they found that cell expressing the TLE6 mutation exhibit impaired TLE6 phosphorylation.

Similarly, through immunoprecipitation and western blot analyses, they further noted that there was reduced binding between OOEP and KDHC3L — two other components of SCMC — and mutant TLE6.

"Taken together, these data reveal two, potentially related, phenotypic mechanisms: reduced potential for PKA-catalyzed phosphorylation and reduced potential to bind SCMC," Alkuraya and his colleagues wrote.

"Although it is unclear whether the patient phenotype is due to the absence of phosphorylation of S510, or its substitution with tyrosine, we note that both PKA-catalyzed phosphorylation and binding to SCMC have been proposed in the literature to explain the critical role of maternal TLE6 in supporting peri-implantation embryogenesis," they added.