A missense in HSF2BP causing primary ovarian insufficiency affects meiotic recombination by its novel interactor C19ORF57/BRME1

The process of gametogenesis is one of the most complex and highly regulated differentiation programs. It involves a unique reductional cell division, known as meiosis, to generate highly specialized cells: the gametes. Indeed, the outcome of meiosis is the production of oocytes and spermatozoa, which are the most distinctive cells of an adult organism and are essential for the faithful transmission of the genome across generations.

The meiotic division is an orderly process that results in the pairing and synapsis of homologous chromosomes and crossover (CO) formation, which ultimately enable homologous chromosomes segregation (Hunter, 2015; Loidl, 2016; Zickler and Kleckner, 2015). In mammals, pairing of homologs is dependent on the repair of self-induced double-strand breaks (DSBs) during prophase I by homologous recombination (Handel and Schimenti, 2010) and it leads to the intimate alignment of homologous chromosomes (synapsis) through the zipper-like synaptonemal complex (SC) (Cahoon and Hawley, 2016). The SC is a proteinaceous tripartite structure that provides the structural framework for DSBs repair (Baudat et al., 2013), as epitomized by the tight association of the recombination nodules (RNs, multicomponent recombinogenic factories) and the axial elements of the SC (Zickler and Kleckner, 2015).

Meiotic DSBs repair is an evolutionarily conserved pathway that is highly regulated to promote the formation of at least one CO per bivalent. This chromosome connection between bivalents through chiasmata is required for a correct reductional division. As other DNA repair processes, proper meiotic recombination is essential for genome stability and alterations can result in infertility, miscarriage and birth defects (Geisinger and Benavente, 2017; Handel and Schimenti, 2010; Webster and Schuh, 2017).

Infertility refers to failure of a couple to reproduce and affects 10–15% of couples (Isaksson and Tiitinen, 2004). Infertility can be due to female factors, male factors, a combination of both or to unknown causes, each category representing approximately 25% of cases (Isaksson and Tiitinen, 2004; Matzuk and Lamb, 2008). There are several underlying causes and physiological, genetic and even environmental and social factors can play a role. Forward and reverse genetic analyses in model organisms have identified multiple molecular pathways that regulate fertility and have allowed to infer reasonable estimates of the number of protein-coding genes essential for fertility (de Rooij and de Boer, 2003; Schimenti and Handel, 2018).

Primary ovarian insufficiency (POI) is a major cause of female infertility and affects about 1–3% of women under 40 years of age. It is characterized by cessation of ovarian function before the age of 40 years. POI results from a depletion of the ovarian follicle pool and can be isolated or syndromic. Genetic causes of POI account for approximately 20% of cases (Rossetti et al., 2017). Although infertility-causing pathogenic variants are inherently unlikely to spread in a population, they can be observed within families, especially when there is consanguinity. Such cases provide crucial insights into the function of the genes and molecular mechanisms that they disrupt. Over the last decade, causative variants in several genes have been found using whole exome sequencing in ‘POI pedigrees’. In particular, pathogenic variants in genes involved in DNA replication, recombination or repair, such as STAG3, SYCE1, HFM1, MSH5 and MEIOB have been formally implicated in this condition by ourselves and others (Caburet et al., 2014; Caburet et al., 2019a; de Vries et al., 2014; Guo et al., 2017; Primary Ovarian Insufficiency Collaboration et al., 2014).

In this study, we have identified in a consanguineous family with POI the candidate S167L missense variant in HSF2BP, an essential yet poorly studied meiotic gene. HSF2BP encodes an interactor of the heat-shock response transcription factor HSF2 (Yoshima et al., 1998). During the course of this work and, in agreement with our results, two independent groups showed that HSF2BP is essential for meiotic recombination through its ability to interact with BRCA2 (Brandsma et al., 2019; Zhang et al., 2019). Here, we report that the introduction of the missense variant HSF2BP-S167L in mouse leads to subfertility and DNA repair defects during prophase I. In addition, we identified a protein complex composed of BRCA2, HSF2BP, and the as yet unexplored C19ORF57/BRME1 (meiotic double-stranded break BRCA2/HSF2BP complex associated protein) as a key component of the meiotic recombination machinery. Our studies show that a single substitution (S167L) in HSF2BP leads to a reduced loading of both BRME1 and HSF2BP at the RNs. Furthermore, our results suggest that meiotic progression requires a critical threshold level of HSF2BP/BRME1 for the ulterior loading of the recombinases to the RNs.

Using exome sequencing, we identified the S167L missense variant in HSF2BP in a consanguineous family with three cases of POI with secondary amenorrhea. All affected family members are homozygous for the variant, and the healthy relatives are heterozygous carriers. The causality of the HSF2BP-S167L variant is supported by the meiotic phenotype and the subfertility observed in Hsf2bp^S167L/S167L female mice. Furthermore, the DNA repair defects in murine Hsf2bp^S167L/S167L meiocytes, displayed by the reduced number of RAD51/DMC1 foci on DSBs and the subsequent reduction in the number of COs, provide evidence that this missense variant alters meiotic recombination. This conclusion was further supported by the comparative analysis of the S167L allele with the Hsf2bp null allele, which revealed that the missense variant can be considered as a hypomorphic allele. This is in agreement with the secondary amenorrhea observed in the patients, and the residual (medically-assisted) fertility in one of the affected sisters. Our identification of HSF2BP as a gene implicated in POI is in line with recent reports of POI-causing variants in genes that are required for DNA repair and recombination, such as MCM8, MCM9, SYCE1, MSH4, PSMC3IP, FANCM or NBN (AlAsiri et al., 2015; Carlosama et al., 2017; de Vries et al., 2014; Fouquet et al., 2017; He et al., 2018; Tenenbaum-Rakover et al., 2015; Tucker et al., 2018; Wood-Trageser et al., 2014; Zangen et al., 2011).

Meiotic mouse mutants often exhibit sexually dimorphic phenotypes (Cahoon and Libuda, 2019). These differences can have a structural basis, given that the organization of the axial elements is known to be different between sexes. This is supported by the difference in length of the axes and by the essential role that the meiotic cohesin subunit RAD21L plays in males but not in females (Herrán et al., 2011). In general, meiotic recombination mutants appear to proceed further in female than in male, because in males the asynapsis of the sex bivalent leads to a loss of silencing of the Y chromosome, and perhaps also because of the presence of less stringent checkpoints in oogenesis (Hunt and Hassold, 2002). A sexually dimorphic phenotype is also observed here in our HSF2BP-S167L mutant mice. Both sexes show a significant decrease in the number of COs and consequently an increase in the frequency of meiocytes showing bivalents without CO. This leads to a significant reduction of spermatozoa in the epididymis, while the number of oocytes and their distribution in the follicular pool are not affected in females. Our failure to detect any significant impact of the variant on male fertility could be due to the high variability in litter frequency and size in our study. However, it is known that a strong reduction of the spermatozoa count (up to 60%) does not affect male mouse fertility (Schürmann et al., 2002), which would explain the normal fertility of male mice bearing the HSF2BP-S167L variant despite the presence of overt meiotic alterations. By contrast, female mice with the HSF2BP-S167L variant show a mild sub-fertility phenotype with a reduction of litter frequency. This can be due to the very much fewer gametes available for fertilization in females in comparison to males but also to molecular differences in the meiotic recombination process in both sexes (Cahoon and Libuda, 2019), as displayed by the absence of RPA accumulation or the more pronounced decrease in DMC1 foci observed in HSF2BP-S167L oocytes.

Although we cannot exclude that a POI-like phenotype would appear over time in HSF2BP-S167L female mice, the sub-fertility observed in HSF2BP-S167L females appears to be milder in comparison to the phenotype of the human patients. This could be explained by a lower sensitivity of mice to hypomorphic alleles compared to humans, in a similar manner to the known lower gene dosage sensitivity of the former in the context of genes that are haploinsufficient in human (Veitia, 2003). In addition, the initial events of human female meiosis appear to be more error-prone than in mice, or even than human males, as evidenced by the increased incidence of synaptic defects in the human oocytes or the fact that MLH1 foci appear much earlier in prophase I (Hassold et al., 2007). Furthermore, it has recently been shown that human oocytes exhibit a specific CO maturation inefficiency (Wang et al., 2017). Indeed, despite a higher total number of COs in women, the frequency of bivalents without CO is paradoxically higher in women than in men. Altogether, these observations could explain the stronger phenotype of the human POI patients compared to the Hsf2bp^S167L/S167L female mice.

The absence of homozygous male carriers in the consanguineous family studied here prevents the direct comparison of the impact of the HSF2BP-S167L variant on fertility phenotypes between human and mouse males. Although future studies might identify infertile men homozygous for the S167L variant, it is interesting to note that another variant of HSF2BP (G224*) was shown to affect recombination rate in males and that two siblings homozygous for this HSF2BP variant in the analyzed Icelandic population were healthy but without descendants, suggesting they were infertile (Halldorsson et al., 2019). This reinforces a conserved function of HSF2BP in human male fertility.

We have shown through a biochemical analysis that BRME1 immunoprecipitates mainly with its partner HSF2BP (constituting or belonging to a complex) but also with PALB2, RAD51, RPA and BRCA2 (in testis extracts). These interactions could possibly be mediated by the multidomain hub protein BRCA2 (Siaud et al., 2011) as HSF2BP interacts directly with BRCA2 (Brandsma et al., 2019) and with BRME1 (this work). In addition, BRCA2 also directly interacts with the DSBs recruiter PALB2, with the recombinases RAD51 and DMC1 (through different specific domains), and with DNA (Siaud et al., 2011). This BRCA2-containing complex participates in the orderly orchestration of events at DSBs such as the initial binding of RPA to the resected DNA, the exchange of RPA by RAD51/DMC1, and the loading of the MEIOB-SPATA22 complex to the RPA complexes (Martinez et al., 2016; Zhao et al., 2015). Interestingly, genes with recently identified variants in POI patients are implicated in the repair of induced DSBs at the early stages of meiosis and encode BRCA2-interacting factors, such as MEIOB, DMC1 or BRCA2 itself (Caburet et al., 2019a; Caburet et al., 2020; Caburet et al., 2019b; He et al., 2018). This highlights the crucial importance and the high sensitivity of this particular meiotic step, and the hub role of BRCA2 as a tightly regulated platform for correct meiotic recombination.

We have also shown by several complementary approaches that the proteins HSF2BP/BRME1 constitute in vivo a functional complex in which both subunits are essential for meiotic recombination and for their mutual protein expression and/or stability in vivo. Accordingly, the genetic depletion of BRME1 or HSF2BP leads to similar if not identical phenotypes in which oogenesis is altered with severe defects in chromosome synapsis that promotes premature loss of ovarian follicles and spermatogenesis is arrested at zygotene-like stage resulting in a lack of spermatozoa. These meiocytes are not able to load the recombinases RAD51/DMC1, impairing the proper repair of DSBs leading to the generation of no COs or very few in males and females, respectively. As a consequence, zygonema-like spermatocytes accumulate the single strand binding proteins SPATA22 and RPA, whereas oocytes accumulates only SPATA22. During the course of the reviewing of this work, three Brme1 knockouts have been described (Zhang et al., 2020; Shang et al., 2020; Takemoto et al., 2020). All the described male mutants show strong fertility defects and similar molecular alterations although with different severity. In females, the two works that address their analysis (Shang et al. and Takemoto et al.), describe normal fertility which is in contrast with the strong reduction in the follicle pool and meiotic defects observed in our Brme1^-/- females. The higher severity of our male and female mutants could be explained on the basis of the different genetic background of the mice given that all of them are apparently similar.

The S167L human recessive POI variant behave as a hypomorphic allele in mice, which results in a reduction of the protein expression/stability of itself and of its partner BRME1 in vivo and in transfected cells. As a consequence, both male and female Hsf2bp^S167L/S167 mice show a similar but milder phenotype than that of the Hsf2bp^-/-or Brme1^-/-, consisting in a reduction in the spermatozoa count while being fertile and a subtle reduction in female fertility. Molecularly, the reduction observed in the meiocytes of the mutant Hsf2bp^S167L/S167L mice of RAD51/DMC1, the reduction of COs (both in males and females), the accumulation of RPA (only in males) and SPATA22 (in males and females) are also weaker than in the null mutant. The observed accumulation of RPA in males is likely to occur at the early stages of recombination because SPATA22 loading to the DSBs is also increased in the mutants of HSF2BP and BRME1. RPA, as part of a trimeric replication protein complex (RPA1-RPA2-RPA3), binds and stabilizes ssDNA intermediates that form during DNA repair. In meiosis, RPA is also forming a complex with two other essential meiotic players MEIOB (homologue of RPA) and SPATA22. However, the loading of this complex to DSBs is RPA-independent (Shi et al., 2019). It has been postulated that RPA functions in meiosis at two different stages; (i) during the early recombination stages when the DSBs ends are resected by the MRN complex and (ii) during the strand invasion into the homologous duplex that is carried out by RAD51/DMC1 and ssDNA is generated at the displacement loops (Shi et al., 2019). The observed lack of DNA-binding ability of HSF2BP/BRME1 points towards a model in which the absence of the complex HSF2BP/BRME1 through a direct interaction with BRCA2 impairs the replacement of RPA by RAD51/DMC1 in the foci that form on the DSBs of the spermatocytes. Similarly, the reduced expression at the protein level of HSF2BP/BRME1 as a consequence of the POI variant, which does not affect their heterodimerization, would make them less proficient in replacing RPA in the spermatocytes by the recombinases RAD51/DMC1 leading to a lower frequency of COs. Given the unknown function that RPA plays in vivo during oogenesis (Shi et al., 2019), it is tempting to speculate that the role of RPA in mediating the replacement of RAD51/DMC1 in female meiosis would be carried out by another protein complex such as SPATA22/MEIOB in a HSF2BP/BRME1-dependent manner.

Very recently, a high-resolution genome-wide recombination map revealed novel loci involved in the control of meiotic recombination and highlighted genes involved in the formation of the SC (SYCE2, RAD21L, SYCP3, SIX6OS1) and the meiotic machinery itself as determinants of COs (Halldorsson et al., 2019). Within the second category, variants of the SUMO ligase RNF212 and the ubiquitin ligase HEI10 have been largely documented as genetic determinants of the recombination rate in humans and, importantly, so were variants of HSF2BP. Consequently, gene dosage of RNF212 and HEI10 affects CO frequency through their activity in CO designation and maturation (Lake and Hawley, 2013; Reynolds et al., 2013). We found that both BRME1 and HSF2BP localization are unaffected in the loss-of-function mouse mutants of Rnf212 and Hei10 (Ccnb1ip1). This observation together with the proper co-localization of BRME1/HSF2BP with RPA allows us to map these proteins upstream in the recombination pathway.

It is worth noting that some of the genes affecting the recombination rate have also been described as ‘fertility genes’, such as SYCP3, HFM1 and HSF2BP (Geisinger and Benavente, 2017; Primary Ovarian Insufficiency Collaboration et al., 2014 and this work). Altogether, we propose that different variants of the same meiotic gene (alleles responsible for mild or strong phenotypes) can give rise to either an altered genome-wide recombination rate with no detrimental effect, or cause infertility when the decreased recombination rate falls below the lower limit of one COs per bivalent. In the present POI family, the S167L variant in HSF2BP seems to be below that limit. To our knowledge, HSF2BP is one of the very few human genes with variants known to affect both the genome-wide recombination rate in the human population and meiotic chromosome missegregation (fertility) through a reduction of the recombination rate (Halldorsson et al., 2019). Along similar lines, it is conceivable that variants with additive effects (Schimenti and Handel, 2018) can lead to a genome-wide reduction of the recombination rate and thus to aneuploidy and infertility. Specifically, variants in genes involved in meiotic recombination and SC constituents could be responsible for a large fraction of genetic infertilities. These variants should be under purifying selection and would be removed or substantially reduced from the population. However, this is not the case for genes with sexual phenotypic dimorphism (Gershoni and Pietrokovski, 2014) as is apparent for a wide number of meiotic genes (Cahoon and Libuda, 2019), including HSF2BP and BRME1, where individuals of one of the sexes are fertile carriers.

In summary, we describe for the first time a human family where POI co-segregates with a genetic variant in HSF2BP (S167L) in a Mendelian fashion. Humanized mice reveal that the HSF2BP variant is a hypomorphic allele that promotes the lower protein expression and/or stability of the HSF2BP/BRME1 complex and phenocopy in a milder manner the meiotic defects observed in mice lacking either HSF2BP or its direct interactor BRME1.

All data generated or analysed during this study are included in the manuscript and supporting files.

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Author details

1. Natalia Felipe-Medina

   Molecular Mechanisms Program, Centro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer (CSIC-Universidad de Salamanca), Salamanca, Spain

   Contribution

   Resources, Formal analysis, Supervision, Investigation, Methodology, Writing - review and editing

   Contributed equally with

   Sandrine Caburet

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6975-2524

2. Sandrine Caburet

   1. Université de Paris, Paris Cedex, France
   2. Institut Jacques Monod, Université de Paris, Paris, France

   Contribution

   Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review and editing

   Contributed equally with

   Natalia Felipe-Medina

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7404-8213

3. Fernando Sánchez-Sáez

   Molecular Mechanisms Program, Centro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer (CSIC-Universidad de Salamanca), Salamanca, Spain

   Contribution

   Formal analysis, Methodology

   Competing interests

   No competing interests declared

4. Yazmine B Condezo

   Molecular Mechanisms Program, Centro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer (CSIC-Universidad de Salamanca), Salamanca, Spain

   Contribution

   Formal analysis, Methodology

   Competing interests

   No competing interests declared

5. Dirk G de Rooij

   Reproductive Biology Group, Division of Developmental Biology, Department of Biology, Faculty of Science, Utrecht University, Utrecht, Netherlands

   Contribution

   Formal analysis, Investigation

   Competing interests

   No competing interests declared

6. Laura Gómez-H

   Molecular Mechanisms Program, Centro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer (CSIC-Universidad de Salamanca), Salamanca, Spain

   Contribution

   Formal analysis, Methodology

   Competing interests

   No competing interests declared

7. Rodrigo Garcia-Valiente

   Molecular Mechanisms Program, Centro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer (CSIC-Universidad de Salamanca), Salamanca, Spain

   Contribution

   Formal analysis

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0444-5587

8. Anne Laure Todeschini

   1. Université de Paris, Paris Cedex, France
   2. Institut Jacques Monod, Université de Paris, Paris, France

   Contribution

   Formal analysis, Investigation

   Competing interests

   No competing interests declared

9. Paloma Duque

   Molecular Mechanisms Program, Centro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer (CSIC-Universidad de Salamanca), Salamanca, Spain

   Contribution

   Formal analysis, Methodology

   Competing interests

   No competing interests declared

10. Manuel Adolfo Sánchez-Martin

    1. Transgenic Facility, Nucleus platform, Universidad de Salamanca, Salamanca, Spain
    2. Departamento de Medicina, Universidad de Salamanca, Salamanca, Spain

    Contribution

    Resources, Methodology

    Competing interests

    No competing interests declared

11. Stavit A Shalev

    1. The Genetic Institute, "Emek" Medical Center, Afula, Israel
    2. Bruce and Ruth Rappaport Faculty of Medicine, Technion, Haifa, Israel

    Contribution

    Resources

    Competing interests

    No competing interests declared

12. Elena Llano

    1. Molecular Mechanisms Program, Centro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer (CSIC-Universidad de Salamanca), Salamanca, Spain
    2. Departamento de Fisiología y Farmacología, Universidad de Salamanca, Salamanca, Spain

    Contribution

    Conceptualization, Formal analysis, Investigation, Writing - original draft, Writing - review and editing

    Competing interests

    No competing interests declared

13. Reiner A Veitia

    1. Université de Paris, Paris Cedex, France
    2. Institut Jacques Monod, Université de Paris, Paris, France
    3. Université Paris-Saclay, Institut de Biologie F. Jacob, Commissariat à l’Energie Atomique, Fontenay aux Roses, France

    Contribution

    Conceptualization, Funding acquisition, Investigation, Writing - original draft, Writing - review and editing

    For correspondence

    reiner.veitia@ijm.fr

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-4100-2681

14. Alberto M Pendás

    Molecular Mechanisms Program, Centro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer (CSIC-Universidad de Salamanca), Salamanca, Spain

    Contribution

    Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing - original draft, Writing - review and editing

    For correspondence

    amp@usal.es

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-9264-3721

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