ABSTRACT

Aim

Infertility is primarily caused by premature ovarian insufficiency (POI). Since multiple genes have been linked to the genetic foundation of POI, the genetic investigation of the condition needs to be a component of the clinical diagnosis. Analyzing the genetic background of POI in a Turkish cohort was the goal of our investigation.

Materials and Methods

The onset age ranged from 18 to 39 years old. Every patient was prescreened for the most common POI-associated fragile-X premutation premutation and had the karyotype 46,XX. Next-generation sequencing (NGS) of 26 genes previously linked to POI was performed on 68 unrelated individuals from Türkiye in order to detect genetic changes.

Results

We examined the DNA samples of 68 unrelated POI patients in order to use targeted panel sequencing to find possible causal variations of the disease. Three POI-related genes in our sample had three heterozygous variants of unclear significance and one heterozygous potentially pathogenic gene. These variants were related to 3 genes: Newborn ovary homeobox (NOBOX), GDF9 and STAG3.

Conclusion

POI is distinguished by a complex genetic background with an increasing number of genes and diverse phenotypic traits. This is the first genetic epidemiology study in Türkiye focusing on the effects of 26 genes related to POI. Among the variations we detected in our patient group, the variation we detected in the STAG3 gene has not been reported before. Two separate variations were detected in the NOBOX gene in two patients. Finally, one variation was detected in the GDF9 gene. The variation in the STAG3 gene was classified as likely pathogenic. The variations in the NOBOX and GDF6 genes are classified as of unknown clinical significance. Due to the intricate network governing human folliculogenesis, individual patients exhibit significant phenotypic diversity, necessitating the development of NGS sequencing methods to aid in POI diagnosis.

INTRODUCTION

Premature ovarian insufficiency (POI), also referred to as premature ovarian failure, denotes the cessation of ovarian function before the age of 40 years. By the end of the 2000s, the term POI was adopted to describe this condition of premature ovarian ageing more accurately. This terminology highlights that women with this dysfunction may occasionally experience spontaneous follicular development, the return of menses, and even conception following the diagnosis^{1, 2}. POI is characterized by the depletion of ovarian follicles, leading to infertility before the age of 40 years, and presents with a diverse range of clinical phenotypes³. This condition is defined by the absence of menses (amenorrhea or oligomenorrhea) for a minimum of four months, elevated gonadotropin levels [follicle-stimulating hormone (FSH) > luteinizing hormone], and hypoestrogenism⁴. Primary amenorrhea, typically identified early in life in individuals with delayed puberty, absent breast development, and menarche, can be the first warning sign. The most common POI phenotype, secondary amenorrhea, however, manifests between the ages of 20 years and 40 years and is characterized by an irregular menstrual cycle followed by amenorrhea alongside normal pubertal development³. The studies showed that the global overall prevalence of POI among women was 3.5%. By subgroup analysis, the prevalence of POI among women with iatrogenic etiology was 11.2%, followed by autoimmunity (10.5%); the prevalence of POI by region was 11.3% at the highest in North America followed by South America (5.4%); and the prevalence of POI was 5.3% in a developing country, higher than 3.1% in a developed country. The trend of prevalence of POI over the past 20 years was on the rise (although p>0.05)⁵.

Genetic flaws, autoimmune disorders, iatrogenic causes (such as chemotherapy or radiation therapy), viral infections, poisons, or even idiopathic conditions can all contribute to POI⁵.

Nonetheless, many impacted women have a positive family history. POI with various genetic etiologies may suggest a hereditary status of the cases⁶. Surgical, medical, infectious or autoimmune ovarian damages are the other well-known reasons for POI⁷.

Regarding genetic factors, the POI phenotype can be instigated by chromosomal abnormalities and monogenic disorders. Following the confirmation of a clinical diagnosis of POI, chromosome analysis, fragile-X premutation (FMR1) testing, evaluation of thyroid and adrenal (21-hydroxylase) antibodies, and pelvic ultrasonography should all be performed³. Approximately 10-13% of individuals exhibit chromosomal abnormalities⁸. In terms of chromosomal origins, X-chromosome abnormalities account for 12% of POI cases. These abnormalities encompass monosomy, trisomy, deletions, duplications, and X-autosome translocations⁹. Critical X chromosomal regions potentially correlating with POI have been identified through cytogenetic studies. POI has been associated with deletions in the Xq21.3-q27 region or X-autosome translocations in the Xq13.3-q21.1 region. Additionally, POI has been linked to the deletion of the p arm of the X chromosome. Cytogenetic analysis can be utilized to evaluate karyotypes for numerical alterations, and various methods, including array comparative genomic hybridization, have been developed to identify copy number variants within the context of POI¹⁰.

Furthermore, expanding a cytosine-guanine-guanine repeat in the 5’ regulatory region of the FMR1 gene, which results in Fragile-X syndrome, may also contribute to syndromic POI. Since the FMR1 premutation is linked to POI in approximately 20% of affected women, its presence in those diagnosed with POI should be investigated¹¹. Microdeletions in the FMR2 gene may also significantly contribute to POI, as suggested by another study¹². Most cases of POI remain without a clear underlying cause; however, this screening might be useful in identifying the etiology of POI. Genetic disorders involved include not only Turner syndrome and FMR1 gene premutation, but also monogenic disorders. Karyotype analysis is insufficient to resolve all cases of POI due to low resolution. Since FMR1 gene premutation analysis can only resolve some of the cases (3% to 15% of cases of POI), monogenic gene analysis appears to be the most accurate method choice¹³. Therefore, monogenic analysis should be the next step if the tests do not provide any positive findings substantiating the POI diagnosis.

Given that the intricate network governing human folliculogenesis leads to significant phenotypic variance in POI syndrome with a diverse genetic etiology, NGS analysis could offer a more accurate, rapid, and cost-effective genetic diagnosis for POI^14-2. Furthermore, a theory positing oligogenic origins for this condition has been proposed, highlighting the necessity for multigene panel sequencing^{15, 16}. Previous studies suggest that these genes may be clustered in the POI1 and POI2 loci on the female sex chromosome¹⁷. It is suggested that several X- and autosome-encoded genes are critical candidates for POI, as they may play a role in human folliculogenesis. A more effective diagnostic pathway could be developed by further investigating their functional contributions to the genetic etiology of POI in clinical settings.

Our understanding of the genetic causes of idiopathic POI has significantly increased in the Next-generation sequencing (NGS) era. Numerous novel pathogenic variants of well-known genes [FSHR, GDF9, BMP15, FIGLA, and Newborn ovary homeobox (NOBOX)] have been linked to POI through high-throughput sequencing approaches³. Due to their roles in sex chromosome remodeling, metabolism, autoimmune associations, meiosis and DNA repair, oogenesis, folliculogenesis, hormone signaling, and germ cell development, these genes have been proposed to play a part in the etiology of POI. Furthermore, extensive genomic research paves the way for discovering additional gene variants underlying currently unknown POI. Our expanding knowledge may lead to more promising results when analyzing the genetic makeup of POI patients and may unveil new pathways for discovering potential treatments for women with POI. This is the only comprehensive genomic investigation to date on Turkish POI patients that utilizes the potential of the NGS method.

MATERIALS AND METHODS

Subjects

Sixty-eight Turkish-unrelated patients diagnosed with POI, who experienced amenorrhea for at least six months before the age of 40 years and had FSH plasma levels exceeding 40 IU/L, were recruited. The age of onset ranged from 18 to 39 years. Each patient underwent an FMR1 molecular analysis and had at least 20 cells karyotyped. We excluded patients found to have Turner syndrome based on the karyotype or any other karyotype abnormality, as well as patients with a FMR1 premutation. Patients who previously underwent a gonadotoxic treatment (chemotherapy or pelvic radiation) or extensive ovarian surgery were also excluded from the study. NGS analysis was performed after all these exclusion criteria were met. Each patient signed a paper granting informed consent. The local ethics committee approved the study. All procedures performed in the study involving human participants were approved by the Ethics Committee for Scientific Research, Faculty of Medicine, Trakya University (decision no: TÜTF-BAEK 2018/319, date: 01.10.2018) and followed the Declaration of Helsinki. Targeted panel sequencing was performed on 68 POI patients (P01∼P68).

Targeted Panel Sequencing

Our goal has been to cover every known POI risk locus. Based on information from the literature, a list of 26 genes under investigation was compiled (Table 1).

Sixty-eight samples were sequenced using the QIAseq Targeted DNA Custom Panel (Qiagen, Hilden, Germany). Two milliliters (2 mL) of peripheral blood were collected and preserved in anticoagulation tubes. Genomic DNA was isolated from peripheral whole blood using the EZ1 DNA Investigator Kit (Qiagen, Hilden, Germany). After DNA extraction, target sequences were enriched using customized capture probe chips (Illumina, San Diego, CA). This kit included 26 genes associated with the disease. Libraries covering the target genes were prepared according to the QIAseq Targeted DNA Panel Protocol (Qiagen, Hilden, Germany). Following the target enrichment process, libraries were sequenced on the MiSeq system (Illumina, San Diego, CA, USA). Obsessive compulsive inventory analysis (Qiagen, Hilden, Germany) was employed to control quality and generate Variant Call Format files. In silico evaluation of the pathogenicity of nucleotide changes in exons was performed using Polymorphism Phenotyping v2 (PolyPhen-2, http:// genetics.bwh.harvard.edu/pph2/), Sorting Intolerant from Tolerant (SIFT, https://sift.bii.a-star.edu.sg/), and MutationTaster (http://www.mutationtaster.org). Minor allele frequencies were checked in the Genome Aggregation Database gnomAD (http://gnomad.broadinstitute.org/). Variant analysis was conducted using Ingenuity software (Qiagen, Hilden, Germany). Variants were interpreted according to the American College of Medical Genetics and Genomics (ACMG) recommended standard. Sanger sequencing was performed for confirmation when target region coverage was less than 15 reads. Nucleotide alterations were analyzed and validated by Sanger sequencing. After confirmation, each variant was classified as a pathogenic, likely pathogenic, variant of unknown significance (VUS), likely benign, or benign, according to the ACMG guidelines. Coding genomic regions that were sequenced with coverage less than 15X were eventually re-sequenced using Sanger technology. All detected variations were confirmed to be accurate by Senger sequencing.

NGS assays produced ∼14,5 GB data for 68 samples for each individual as pair-end reads, having up to 97 bp mean read length, and about 90% (0.2 Mb length) of the targeted bases were covered, thereby sufficiently passing our thresholds for calling single nucleotide polymorphisms and short insertions or deletions (indels).

Primer sets were created for all required areas in order to execute Sanger sequencing on an ABI 3130 (Applied Biosystems, USA) capillary electrophoresis machine and validate the variations and segregation analyses.

RESULTS

We examined the DNA samples of sixty-eight unrelated patients with POI using targeted panel sequencing to identify potential causal variations of the disease. Within our sample, three POI-related genes exhibited three heterozygous VUS and one heterozygous gene that may be pathogenic. With the assistance of Franklin (available online at https://franklin.genoox.com) and VarSome¹⁸, we detailed the clinical interpretation and implications for the identified variants. All variants identified were heterozygous, with most classified as missense (Table 2).

A total of one likely pathogenic variant (STAG3) was identified in 1.47% (1 of 68) of POI patients, which is regarded as a molecular genetic diagnosis of POI. VUS were detected in two genes in three patients from the total patient group. The genes identified were NOBOX and GDF9. A frameshift was recognized in one of the 68 patients.

Statistical Analysis

Characteristics of patients were gathered through a review of electronic medical records. Descriptive statistics, such as medians, ranges, and frequencies, were employed.

DISCUSSION

POI is characterized by a robust genetic background with increasing genes and diverse phenotypic traits³. The POI phenotype is associated with detrimental gene variations affecting meiosis, DNA repair, gonadal development (oogenesis and folliculogenesis), hormone signaling, immunological function, and metabolism³. This study represents, to our knowledge, the first genetic epidemiology study in Türkiye focusing on 26 genes in a single panel associated with POI, and several genetic alterations have been identified in genes associated with this condition. Sixty-eight Turkish patients with a clinical diagnosis of POI, who had undergone prescreening for FMR1 pathogenic expansion, were included in our investigation.

The NM_001282717.2 (STAG3):c.1237_1238insAA variation identified in one of our patients constitutes a frameshift alteration that has not previously been reported for POI.

The meiosis-specific subunit of the cohesin complex, encoded by STAG3, comprises SMC1α/SMC1β, SMC3, RAD21/REC8, or RAD21L¹⁹. Sister chromatids are held together during mitosis and meiosis by the multiprotein cohesin complex encircles them in a ring. SMC1β, REC8, and RAD21L are exclusively present in meiotic cells, while SMC1α and RAD21 are ubiquitously found²⁰. Although STAG3 is restricted to the testes and ovaries^{21, 22}, STAG1/2 is present in mitotic cells²³. The phenotypic effect of the STAG3 gene exhibits recessive inheritance.

In the study by Caburet et al.²², the identified homozygous STAG3 variant leads to a premature stop codon, and autosomal recessive inheritance was demonstrated in four sisters of consanguineous parents. In another more recent study, a homozygous donor splice-site variant of STAG3 was reported leading to POI in two females²⁵.

There was no anomaly other than POI in our patient, for whom we detected STAG3 heterozygous frameshift variation. However, the contribution of variations in the STAG3 gene to the POI has been reported in the literature. Our finding is consistent with this finding. However, due to the recessive inheritance of STAG3, we do not expect any phenotypic effects^{13, 26}.

Variations in the NOBOX gene were identified in two patients, both classified as VUS. The NOBOX gene is believed to be one of the primary genetic contributors to POI^{27, 28}. This ovarian homeobox gene is involved in the early stages of folliculogenesis, with the phenotypic effect of the NOBOX gene exhibiting dominant inheritance. Although the variant detected in our patient (rs749172175) is defined as VUS according to ACMG criteria, it is evaluated as “pathogenic” in variant evaluation prediction tools and the allele frequency given as 0.00000881 in the gnomAD database supports this possibility. The variations we detected in the NOBOX gene are consistent with the studies in the literature. Another variation detected in NOBOX, c.1788G > C (p.(Trp596Cys), was evaluated as VUS. However, there are values ​​in the databases suggesting pathogenicity (SIFT: Damaging, CADD (phred): 27.10). However, since both variations were reported as VUS, their clinical significance is unknown²⁹.

A variation classified as a VUS was identified in the GDF9 gene in one of our patients. Clinical evaluation of the patient revealed POI. According to Qin et al.²⁸ and Jiao et al.³⁰, variations in GDF9 are frequently cited among the top 20 common genetic causes of POI. Notably, various GDF9 variations have been found globally, albeit with a slightly uneven distribution by geography and ethnicity. The phenotypic effect of the GDF9 gene exhibits recessive inheritance. Since the GDF9 variation in our patient was both heterozygous and classified as a VUS, we did not consider it likely to have a phenotypic effect. The c.1297G>A variation detected in GDF9 has not been reported before. Therefore, a comparison with the literature could not be made.

High-throughput methods have been essential for identifying novel variants in candidate genes and genes previously linked to POI. The encoded proteins’ primary functions are meiosis and DNA repair, gonadal development (oogenesis and folliculogenesis), hormone signaling, immunological response, and metabolism. As shown in several animal models, changes in genes related to meiosis and DNA repair may result in distinct symptoms of ovarian insufficiency because of the resting state of oocytes³¹.

Study Limitations

There are certain limitations in our study. One of them is the number of the patients. There may not be enough power to detect rare variants with a frequency of less than 5% in 68 patients. We could obtain more results with an increased number of patients. A setting of risk prediction, routine diagnosis, and early intervention will benefit greatly from targeted panels of hotspot mutations and/or validated causal genes, as well as broader analysis like whole-exome and whole genome sequencing in the future.

CONCLUSION

POI is a highly diverse disorder that may have a complex array of genetic variants due to its many contributing causes. Furthermore, phenotypic variability is complicated by the unclear definition and nomenclature of POI, which exacerbates the genetic heterogeneity of POI and vice versa. It is uncertain if different phenotypes-like ovarian dysgenesis, primary or secondary amenorrhea, or early or late onset POI-share the same genetic makeup but have differing cumulative effects. A very large number of variations with unclear significance will surface in the NGS era. Data analysis and filtering will be quite difficult but crucial. Mutation-directed transgenic models or transformation experiments should be used to confirm causal relevance. However, it is getting more and harder to identify a single genetic alteration in any one POI patient as “causative” because many of the known POI genes appear to work in concert while exhibiting characteristics of incomplete penetrance or variable expressivity on their own. The relationship between genetics and phenotype requires more investigation.

Determining who is at risk for POI is still difficult. The potential of NGS technologies in clinical practice is huge.