USP26: a genetic risk factor for sperm X‐Y aneuploidy

Abstract

Segregation of the largely non‐homologous X and Y sex chromosomes during male meiosis is not a trivial task, because their pairing, synapsis, and crossover formation are restricted to a tiny region of homology, the pseudoautosomal region. In humans, meiotic X‐Y missegregation can lead to 47, XXY offspring, also known as Klinefelter syndrome, but to what extent genetic factors predispose to paternal sex chromosome aneuploidy has remained elusive. In this issue, Liu et al (2021) provide evidence that deleterious mutations in the USP26 gene constitute one such factor.

Analyses of Klinefelter syndrome patients and Usp26‐deficient mice have revealed a genetic influence on age‐dependent sex chromosome missegregation during male meiosis.

Multilayered mechanisms have evolved to ensure successful X‐Y recombination, as a prerequisite for subsequent normal chromosome segregation. These include a distinct chromatin structure as well as specialized proteins on the pseudoautosomal region (Kauppi et al, 2011; Acquaviva et al, 2020). Even so, X‐Y recombination fails fairly often, especially in the face of even modest meiotic perturbations. It is perhaps not surprising then that X‐Y aneuploidy—but not autosomal aneuploidy—in sperm increases with age (Lowe et al, 2001; Arnedo et al, 2006), as does the risk of fathering sons with Klinefelter syndrome (De Souza & Morris, 2010).

Klinefelter syndrome is one of the most common aneuploidies in liveborn individuals (Thomas & Hassold, 2003). While most human trisomies result from errors in maternal chromosome segregation, this is not the case for Klinefelter syndrome, where the extra X chromosome is equally likely to be of maternal or paternal origin (Thomas & Hassold, 2003; Arnedo et al, 2006). Little is known about genetic factors in humans that predispose to paternal XY aneuploidy, i.e., that increase the risk of fathering Klinefelter syndrome offspring. The general notion has been that paternally derived Klinefelter syndrome arises stochastically. However, fathers of Klinefelter syndrome patients have elevated rates of XY aneuploid sperm (Lowe et al, 2001; Arnedo et al, 2006), implying a persistent defect in spermatogenesis in these individuals rather than a one‐off meiotic error.

To identify possible genetic factors contributing to Klinefelter syndrome risk, Liu et al (2021) performed whole‐exome sequencing in a discovery cohort of > 100 Klinefelter syndrome patients, followed by targeted sequencing in a much larger cohort of patients and controls, as well as Klinefelter syndrome family trios. The authors homed in on a mutational cluster (“mutated haplotype”) in ubiquitin‐specific protease 26 (USP26), a testis‐expressed gene located on the X chromosome. Effects of this gene’s loss of function (Usp26‐deficient mice) on spermatogenesis have recently been independently reported by several laboratories and ranged from no detectable fertility phenotype (Felipe‐Medina et al, 2019) to subfertility/sterility associated with both meiotic and spermiogenic defects (Sakai et al, 2019; Tian et al, 2019). With their Klinefelter syndrome cohort findings, Liu et al (2021) also turned to Usp26 null mice, paying particular attention to X‐Y chromosome behavior and—unlike earlier mouse studies—including older mice in their analyses. They found that Usp26‐deficient animals often failed to achieve stable pairing and synapsis of X‐Y chromosomes in spermatocytes, produced XY aneuploid sperm at an abnormally high frequency, and sometimes also sired XXY offspring. Importantly, these phenotypes only occurred at an advanced age: XY aneuploidy was seen in six‐month‐old, but not two‐month‐old Usp26‐deficient males. Moreover, levels of spindle assembly checkpoint (SAC) proteins also reduced in six‐month‐old males. Thus, in older Usp26 null mice, the combination of less efficient X‐Y pairing and less stringent SAC‐mediated surveillance of faithful chromosome segregation allows for sperm aneuploidy, providing another example of SAC leakiness in males (see Lane & Kauppi, 2019 for discussion).

Liu et al’s analyses shed some light on what molecular mechanisms may be responsible for the reduced efficiency of X‐Y pairing and synapsis in Usp26‐deficient spermatocytes. USP26 codes for a deubiquitinating enzyme that has several substrates in the testis. Because USP26 prevents degradation of these substrates, their levels should be downregulated in Usp26 null testes. Liu et al (2021) show that USP26 interacts with TEX11, a protein required for stable pairing and normal segregation of the X and Y chromosomes in mouse meiosis (Adelman & Petrini, 2008). USP26 can de‐ubiquitinate TEX11 in vitro, and in Usp26 null testes, TEX11 was almost undetectable. It is worth noting that USP26 has several other known substrates, including the androgen receptor (AR), and therefore, USP26 disruption likely contributes to compromised spermatogenesis via multiple mechanisms. For example, AR signaling‐dependent hormone levels are misregulated in Usp26 null mice (Tian et al, 2019).

The sex chromosome phenotypes observed in Usp26 null mice predict that men with USP26 mutations may be fertile, but producing XY aneuploid sperm at an abnormally high frequency, and that spermatogenic defects should increase with age (Fig 1). These predictions were testable, because the mutated USP26 haplotype, present in 13% of Klinefelter syndrome patients, was reasonably common also in fertile men (7–10%). Indeed, sperm XY aneuploidy was substantially higher in fertile men with the mutated USP26 haplotype than in those without USP26 mutations. Some mutation carriers produced > 4% aneuploid sperm. Moreover, age‐dependent oligospermia was also found associated with the mutated USP26 haplotype.

Figure 1. Mutated USP26 as genetic risk factor for age‐dependent X‐Y defects in spermatogenesis.

Mouse genetics demonstrate that deleterious USP26 mutations lead to less‐efficient X‐Y pairing and recombination with advancing age. Concomitant decrease of spindle assembly checkpoint (SAC) protein levels leads to less‐efficient elimination of metaphase I spermatocytes that contain misaligned X and Y chromosomes. This allows for the formation of XY aneuploid sperm in older individuals and subsequently increased age‐dependent risk for fathering Klinefelter syndrome (KS) offspring, two correlates also observed in human USP26 mutation carriers. At the same time, oligospermia/subfertility also increases with advanced age in both Usp26‐deficient mice and USP26 mutation‐carrying men, tempering Klinefelter syndrome offspring risk but also decreasing fecundity.

As indicated by its prevalence in the normal control population, the USP26 mutated haplotype is not selected against in the human population. With > 95% of sperm in USP26 mutation carriers having normal haploid chromosomal composition, the risk of producing (infertile) Klinefelter syndrome offspring remains modest, likely explaining why USP26 mutant alleles are not eliminated. Given that full Usp26 disruption barely affects fertility of male mice during their prime reproductive age (Felipe‐Medina et al, 2019; Tian et al, 2019; Liu et al, 2021), there is little reason to assume strong negative selection against USP26 variants in humans.

USP26 as the first‐ever genetic risk factor predisposing to sperm X‐Y aneuploidy and paternal origin Klinefelter syndrome offspring in humans, as uncovered by Liu et al, may be just one of many. 90% of Liu et al’s Klinefelter syndrome cases were not associated with USP26 mutations. But even in the age of genomics, discovery of Klinefelter syndrome risk factors is not straightforward, since most sperm of risk mutation carriers will not be XY aneuploid and thus not give rise to Klinefelter syndrome offspring. In addition, as Usp26 null mice demonstrate, both genetic and non‐genetic modifiers impact on penetrance of the XY aneuploidy phenotype: Spermatogenesis in the absence of Usp26 was impaired in the DBA/2 but not the C57BL/6 mouse strain background (Sakai et al, 2019), and in older mice, there was substantial inter‐individual variation in the severity of the X‐Y defect (Liu et al, 2021). In human cohorts, genetic and non‐genetic modifiers are expected to blur the picture even more.

Future identification of sex chromosome aneuploidy risk factors has human health implications beyond Klinefelter syndrome. Firstly, XXY incidence is not only relevant for Klinefelter syndrome livebirths—it also contributes to stillbirths and spontaneous abortions, at a 4‐fold higher rate than to livebirths (Thomas & Hassold, 2003). Secondly, persistent meiotic X‐Y defects can, over time, result in oligospermia and even infertility. Since the mean age of first‐time fathers is steadily rising and currently well over 30 years in many Western countries, age‐dependent spermatogenic defects will be of ever‐increasing clinical relevance.

The EMBO Journal (2021) 40: e108552.