Whole exome sequencing used to identify 6 genes involved in recurrent euploid miscarriage

Researchers in a 2023 study discovered 6 genes associated with recurrent miscarriage of euploid embryos, which were found to impair reproductive ability in mouse models.

Euploid embryos can fail for a number of reasons, including due to an underlying infection (endometritis), anatomical abnormalities (fibroids), or immunological issues, and this can lead to recurrent miscarriage. Additionally, there may be mutations in key genes that are needed for embryo development that are not detected by PGT-A.

You can read more about these different reasons in my post Why do embryos in IVF fail to implant or miscarry?

To identify genes that may be involved in recurrent miscarriage, a DNA sequencing technique called whole exome sequencing can be performed. This is different from sequencing the genome (total DNA of an individual). The exome is only about 1.5% of the genome, and corresponds to only protein-coding genes. Whole exome sequencing is generally cheaper than whole genome sequencing, and can identify genes that are mutated by comparing the sequence to a reference genome that doesn’t contain mutations.

Wang et al. (2023) performed whole exome sequencing on 8 couples with recurrent miscarriage. These couples experienced at least 3 historical consecutive miscarriages, as well as miscarriages using PGT-A tested euploid embryos. The products of conception from one of the miscarried euploids was also tested. The study took place between 2014 and 2017 at a IVF center in China.

Key information:

All women were under 36.
They didn’t have any uterine abnormalities (like fibroids or polyps).
The couple had normal karyotypes.
The women didn’t have thrombophilia, diabetes, hypothyroidism or an autoimmune disease.
They hadn’t been diagnosed with an infectious disease within 3 months.
Endometrial thickness was at least 7 mm.

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Whole exome sequencing reveals six genes involved in recurrent miscarriage

Performing whole exome sequencing on the 8 couples and the miscarriage tissue revealed 6 affected genes: ATP2A2, NAP1L1, RYR2, PLXNB2, SSPO and NRK. In most cases, the mutation was inherited from one of the parents, however there were some instances where the mutation occurred spontaneously (de novo) and wasn’t present in the parents.

ATP2A2 (sarcoplasmic/endoplasmic reticulum calcium ATPase 2) is a gene involved in muscle contractions. Previous research in zebrafish have shown that mutation of this gene is lethal in embryos.

RYR2 (Ryanodine receptor 2) is a gene involved in heart muscle cells, and is needed for heart development.

NAP1L1 (Nucleosome assembly protein 1-like 1) is a protein involved in packaging DNA into structures called nucleosomes. This type of protein helps to pack about 2 meters worth of DNA (the physical length of our genome) into our microscopic cells. Previous studies have shown that this gene is also important for the development of the nervous system in the embryo.

PLXNB2 (Plexin-B2) is a receptor protein found in neurons and is important in the development of the nervous system in embryos.

NRK (Nik-related protein kinase) is not well characterized, but may be involved in regulating the development of the placenta.

SSPO (Subcommissural organ-spondin) is involved in the development of the nervous system.

Mutations in RYR2 and PLXNB2 lead to reduced reproductive ability in mice

At this point the researchers had identified these 6 genes that were mutated in the couples and the miscarriage tissue. However, they didn’t know that these mutations caused the miscarriage, so they needed a way to test this.

To do this, they generated mouse models that had mutations in RYR2 and PLXNB2 and mated them to see if they had any problems with reproduction. The mice were otherwise completely normal, so if there were any problems they knew it would be attributed to the mutations they introduced.

They chose to focus on just these two genes because making these mouse models is a lot of work. They chose RYR2 and PLXNB2 because the mouse and human versions of the gene are similar in sequence. Additionally, RYR2 has also been implicated in stillbirths, and PLXNB2 is involved in multiple cellular functions (so mutating it will likely cause embryo lethality).

To make these mice with these mutant genes, they used CRISPR/Cas9 (pronounced “crisper”). This technique allows the researchers to make specific mutations in targeted genes with high accuracy. They used CRISPR to make mutations in the single cell of a fertilized mouse egg, which then went on to form the adult that could be mated and evaluated for reproductive issues.

Because the mutations may be lethal to the embryo and they would never develop, they had to make heterozygotes. Heterozygotes are organisms that have two different versions of a gene. Mice, like humans, are diploid and contain two copies of each of their chromosomes (and therefore two copies of each gene). One copy of the gene was made mutant with CRISPR, while the other was left alone. This way the mouse embryo could survive to develop into an adult, but after mating the heterozygotes some of the offspring would inherit two mutant genes and presumably die.

Now that they had their mutant mice, they bred them together to see how many pups they would make in a litter.

It turns out that mice with the mutant RYR2 or PLXNB2 produced less pups in a litter compared to mice that didn’t have these mutations. This suggests that these genes can impair the reproductive ability of mice.

All of this is one piece of evidence — these two genes can reduce mouse reproductive potential. However this is really just showing subfertility in these mice, and isn’t demonstrating that these mutations are lethal to the mouse embryos. To do that they needed to do more studies on the actual embryos, which they unfortunately didn’t.

Regardless, this helps to put together this idea that these genes may in fact be involved in leading to the recurrent miscarriage that was seen in the patients.

Screening in other euploid miscarriage samples found mutations in RYR2 and PLXNB2

To see if these findings could relate to other euploid miscarriages, the researchers screened 113 euploid miscarriage samples for mutations in RYR2 and PLXNB2. All the samples were from patients who were between 22 and 42 years old.

They found the same type of mutations in RYR2 and PLXNB2 as they did previously, along with an additional 10 new variants in the samples. These variants represent different mutations in RYR2 and PLXNB2 from the ones they first discovered.

Some of the variants were predicted to be at a slightly higher or higher frequency than the East Asian population (representing the individuals in this study), while others were predicted to be rare.

Conclusions

This study found 6 genes that were mutated in couples with recurrent miscarriage: ATP2A2, NAP1L1, RYR2, PLXNB2, SSPO and NRK.

To see if some of these genes can actually lead to impaired reproductive ability, the researchers generated mice with mutations in RYR2 and PLXNB2 using CRISPR. Breeding these mice resulted in fewer pups per litter compared to normal mice, suggesting that RYR2 and PLXNB2 can indeed impair reproductive potential.

The researchers also screened an additional 113 euploid miscarriage samples for mutations in RYR2 and PLXNB2, and found the same mutations in some samples, as well as different variants.

This study shows that whole exome sequencing of couples and euploid miscarriage samples can provide valuable insight into the underlying causes of recurrent miscarriage. It may also lead to the discovery of new genes involved in recurrent miscarriage and facilitate the development of screening strategies and therapeutic interventions.

Reference

Wang X, Shi W, Zhao S, Gong D, Li S, Hu C, Chen ZJ, Li Y, Yan J. Whole exome sequencing in unexplained recurrent miscarriage families identified novel pathogenic genetic causes of euploid miscarriage. Hum Reprod. 2023 May 2;38(5):1003-1018. doi: 10.1093/humrep/dead039. PMID: 36864708; PMCID: PMC10152170.

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About Embryoman

Embryoman (Sean Lauber) is a former embryologist and the founder of Remembryo, an IVF research and fertility education website. After working in an IVF lab in the US, he returned to Canada and now focuses on making fertility research more accessible. He holds a Master’s in Immunology and launched Remembryo in 2018 to help patients and professionals make sense of IVF research. Sean shares weekly study updates on Facebook, Instagram, and Reddit regularly. He also answers questions on Reddit or in his private Facebook group.