Biallelic Mutations in PATL2 Cause Female Infertility Characterized by Oocyte Maturation Arrest

Abstract

Oocyte maturation arrest results in female infertility, but the genetic determinants of human oocyte maturation arrest remain largely unknown. Previously, we identified TUBB8 mutations responsible for human oocyte maturation arrest, indicating the important role of genetic factors in the disorder. However, TUBB8 mutations account for only around 30% of individuals with oocyte maturation arrest; thus, the disorder is likely to involve other genetic factors that are as yet unknown. Here, we initially identified a homozygous nonsense mutation of PATL2 (c.784C>T [p.Arg262^∗]) in a consanguineous family with a phenotype characterized by human oocyte germinal vesicle (GV) arrest. Subsequent mutation screening of PATL2 in a cohort of 179 individuals identified four additional independent individuals with compound-heterozygous PATL2 mutations with slight phenotypic variability. A genetic burden test further confirmed the genetic contribution of PATL2 to human oocyte maturation arrest. By western blot in HeLa cells, identification of splicing events in affected individuals' granulosa cells, and immunostaining in affected individuals' oocytes, we provide evidence that mutations in PATL2 lead to decreased amounts of protein. These findings suggest an important role for PATL2 mutations in oocyte maturation arrest and expand our understanding of the genetic basis of female infertility.

Keywords: Mendelian disease; female infertility; mutations; oocyte maturation arrest.

Figure 1

Identification of Mutations in PATL2 (A) Five pedigrees affected by oocyte maturation arrest. All five affected individuals have biallelic mutations with a recessive inheritance pattern. The proband from family 1 (with consanguineous parents) has a homozygous nonsense mutation in PATL2. The other individuals from families 2–5 carry compound-heterozygous variants derived from both parents. Sanger sequencing confirmation is shown below the pedigrees. Equal signs indicate infertility, and the double line between individuals I-1 and I-2 in family 1 indicates consanguinity. Black circles represent the proband. In family 2, the sister of the affected individual carrying a heterozygous mutation was not married, and her fertility was unknown. (B) Homozygosity mapping of individual II-1 in family 1. Homozygous regions harboring the strongest signal are indicated in red. The asterisk indicates the area where PATL2 is located. (C) Locations and conservation of mutations in PATL2. The positions of all mutations are indicated in the genomic structure of PATL2. The affected amino acids were compared among six mammalian species in a conservation analysis.

Figure 2

Phenotypes of Oocytes Retrieved from Affected Individuals (A) The morphology of normal and affected individuals’ germinal vesicle (GV), metaphase I (MI), and first polar body (PB1) oocytes. Individuals from families 3 and 4 had some PB1 oocytes, but these had abnormal PB1s, as indicated by the arrows. The scale bar represents 50 μm. (B) Immunolabeling of normal and affected individuals’ oocytes at different stages. Oocytes were immunolabeled with antibodies against PATL2 (shown in green) for visualization of the protein distribution and were counterstained with Hoechst 33342 (shown in blue) for DNA visualization. The morphologies of the oocytes were examined with an inverted microscope system (OLYMPUS IX71), and the immunolabeling was examined by confocal microscopy (Leica). PATL2 was mostly located in the cytoplasm of normal oocytes and gradually degraded during oocyte maturation. The affected individuals’ oocytes showed lower fluorescence than the normal oocytes, implying that the amount of PATL2 was greatly impaired by these mutations. The scale bar represents 50 μm.

Figure 3

A Schematic Representation of the PATL2 Splice Aberrations That Lead to Protein Truncation in Affected Individuals (A) Schematic illustrating the effect of the PATL2 c.223−14_223−2del CCCTCCTGTTCCA splice-site mutation. The acceptor −2A deletion disrupted the canonical acceptor splicing site and resulted in an alternative acceptor ahead of the site, causing a 49-bp insertion between exons 3 and 4. (B) Schematic illustrating the effect of the PATL2 c.1224+2T>C splice-site mutation. The donor +2T>C mutation missed the canonical donor splice site and made the next downstream splice site the donor site, causing a 43-bp insertion between exon 12 and exon 13 (transcript variant 1) or exon 14 (transcript variant 2). Both types of splicing mutations resulted in premature termination.