In the present study, using a self-designed target panel covering 28 known POI causative genes screened in 500 Chinese Han patients, 14.40% (72/500) of patients were diagnosed with at least one pathogenic variant contributing to ovarian insufficiency. A total of 58 potential causative variants, including digenic heterozygous variants in MSH4 and MSH5, were firstly reported in POI, which not only expanded the variant spectrum of human POI, but also enriched the genetic architecture of POI pathogenesis.

Under most circumstances, the pleiotropic genes responsible for POI cause syndromic POI, which manifests with highly variable somatic abnormalities in addition to reproductive phenotypes, such as BLM for Bloom syndrome and WRN for Werner syndrome [3]. Recent genetic studies have revealed that variants in pleiotropic genes also resulted in isolated POI, such as NBN and EIF2B2, which could be explained by specific mutation sites and different types of variants [14]. In the present study, variants in pleiotropic genes, including FOXL2, NR5A1, and BMPR2, were identified in patients presenting with isolated POI, confirming that specific variants might contribute to distinct phenotypes of POI. These findings also highlighted the necessity of individualized genetic counseling and long-term healthcare follow-up in these women.

FOXL2 is preferentially expressed in the ovary, eyelids, and pituitary gland [15, 16]. Heterozygous intragenic variants of FOXL2 accounted for 71% of patients with blepharophimosis-ptosis-epicanthus inversus syndrome (BPES), which is a dominant condition characterized by eyelid and mild craniofacial defects associated with POI (type I) or not (type II) [17]. Although more than 100 variants of FOXL2 have been found in BPES patients [17], the constitutional variants were reported in only 1.0%–2.9% of non-syndromic POI cases [18]. Through the panel test, we found that the prevalence of FOXL2 variants in isolated POI was 3.2%, which was much higher than the other genes in the panel. Intriguingly, the variant p.R349G in FOXL2 was firstly reported here and accounted for 2.6% of the cohort, which was significantly higher than the frequency in public databases. FOXL2 is involved in ovarian development by regulating the transcription of essential genes involved in steroidogenesis, including CYP17A1 and CYP19A1 [19,20,21,22]. Functional studies demonstrated that variant p.R349G impaired the transcriptional regressive effect of FOXL2 on CYP17A1, which might further influence the synthesis of estradiol and lead to folliculogenesis abnormalities [21]. Recently, somatic mutation p.C134W in FOXL2 has been found associated with GCs tumor in adult and accounted for up to 5% of ovarian malignancies [23]. Therefore, although none of the FOXL2 variation carriers in our cohort presented with eyelid malformation or ovarian tumor, the long-term follow-up is still warranted.

NR5A1 is a nuclear receptor that regulates the transcription of genes required for adrenal and reproductive development [24]. Variants in NR5A1 are associated with different reproductive phenotypes in humans, such as disorders of sex development (DSD), hypospadias, and POI. It has been reported that 0.3%–2.3% of POI patients carried NR5A1 mutations [25, 26], which is similar to the frequency in our study (1.2%). Interestingly, the variation p.R313C, locating at ligand-binding domain of NR5A1, was one of the most common variants identified in DSD patients [13]. However, the carriers of p.R313C and p.R313H in our study had normal female external genitalia, which might be explained by genetic heterogeneity during gonad differentiation.

BMPR2 is one of bone morphogenetic protein (BMP) binding soluble factors, participating in signal transduction between oocytes and GCs, which is essential for oocyte maturation [27, 28]. Most BMPR2 variants were reported previously in patients with idiopathic pulmonary arterial hypertension (IPAH), while recent NGS and functional studies have revealed that p.S987F in BMPR2 caused isolated POI by perturbing BMP15/BMPR2/SMAD signaling and GCs proliferation [29]. In this study, five variants in BMPR2 were found for the first time in seven patients with POI. It was reported that the majority of the BMPR2 variants identified in POI patients were located in cytoplasmic tail (amino acids 504–1038) of BMPR2 [30, 31]. However, three out of the five variants identified here were localized in kinase domain (amino acids 203–503). Similarly, a recent study also identified a novel heterozygous variant of POI patient in BMPR2 kinase domain (p.Val453Met) [32], suggesting that variants located in different domains of BMPR2 may have individual effects on ovarian function, highlighting the contribution of BMP signal pathway in isolated POI pathogenesis. Although no clinical features of pulmonary hypertension had been found in the time of investigation, the long-term status should be followed.

The inheritance pattern of POI includes recessive, dominant, and X-linked modes. With the accumulation of variants identified by WES and NGS, more complex inheritance patterns have been discovered. In this study, we identified a compound heterozygous variant in NOBOX, a previous dominant pathogenic gene, and digenic heterozygous variants in MSH4 and MSH5 that had never been reported in POI. Our findings provided new insights into the complexity of POI genetics.

NOBOX is an oocyte-specific transcriptional factor that plays a critical role in early folliculogenesis. Heterozygous NOBOX variants can explain up to 6.2% of POI patients via a dominant negative effect or haploinsufficiency [33]. The mutation prevalence of NOBOX in our cohort was 1.2% (6/500). Additionally, one compound heterozygous mutation p.R355H and p.L558fs was found in two POI patients in pedigree F254. The variant p.R355H was proved to disrupt the transcriptional function of NOBOX [10], while the variant p.L558fs results in a truncated NOBOX protein lacking the C-terminal 133 amino acids. However, the proband’s mother with p.R355H mutation presented with normal menstruation cycles and menopause occurred at 48 years of age. It might be explained by an incomplete penetrance effect of the causal variant. Therefore, more evidence is needed to prove the pathogenicity of heterozygous variants in NOBOX for POI.

Genes involved in meiosis are critical for early follicular development. To date, majority of mutations in meiotic genes have been found in biallelic state (homozygous or compound heterozygous), such as HFM1, BRCA2, and STAG3 [7]. MSH4 and MSH5 belong to the DNA mismatch repair gene family. The MSH4-MSH5 heterodimer plays an important role in homologous recombination repair of DNA double strand breaks, which is essential for meiosis [3]. WES in POI pedigrees has identified two homozygous variants in MSH4 and MSH5 previously [34, 35]; however, the contribution of MSH4 or MSH5 variants in the pathogenesis of sporadic POI has not been reported yet. In the present study, one homozygous variant in MSH5 and three compound heterozygous variants in MSH4 inherited in recessive pattern were identified in 5 patients, accounting for 1.0% (5/500) of patients with sporadic POI. Interestingly, patient POI-9 carried digenic heterozygous variants in MSH4 and MSH5, indicating that not only one subunit deficiency, but also dysfunctional MSH4-MSH5 interaction or cumulative haploinsufficiency of both subunits, may disrupt homologous recombination during meiosis, finally causing POI. This is the first report about digenic heterozygous variants occurred in MSH4-MSH5 heterodimer, which sheds new light on the complex genetic architecture of POI and suggests a novel mechanism of POI pathogenesis.

Previous NGS studies showed that 36%-42% of POI patients carried two or more variants in distinct genes [6, 7]. It is speculated that accumulated genetic defects or deleterious environmental exposures might aggravate the insufficient formation or accelerate the exhaustion of oocytes, resulting in diverse severity of POI phenotype. In general, menarche occurs depends on a maturing hypothalamic-pituitary-ovarian (HPO) axis. The insufficient ovarian function presents with delayed or diminished response to pituitary hormones. In this study, compared to the patients with monogenic variants, the 9 patients (1.8%) carrying digenic or multigenic variants tended to exhibit delayed age at menarche, earlier age of POI onset, and greater prevalence of primary amenorrhea. However, the above differences did not reach statistical significance, which may be due to the limitation of small sample size. Similarly, a recent study suggested that the most severe phenotypes were associated with either the major number of variations or a worse prediction in pathogenicity of variants [8]. To a certain extent, our results partially indicated that oligogenic should be considered when affected women in a family present with different phenotypes or diverse severities of POI.

One of the strengths of the present study is the largest cohort of POI patients included. Another strength is that all the candidate genes have reported evidence of confirmative pathogenicity to human POI, and that the criteria for pathogenic and likely pathogenic used to define causative variants is more strict, which is also the possible explanation for relatively lower variant frequency compared to previous studies (14.4% vs. 19% ~ 48%). There were also a few limitations. First, although the identified variants were checked in the public population data, sequencing of control women from Chinese descent is lacking. Second, the coverage of this panel did not capture all known POI-related genes. Finally, not all family members were available for co-segregation analysis or tracing variants initiation.