<figure><img src="image_1.png" /></figure>

Short Communication

# Revisiting *GDF9* variants in primary ovarian insufficiency: A shift from dominant to recessive pathogenicity?

Pénélope Jordan<sup>a,1</sup>, Camille Verebi<sup>a,1</sup>, Bérénice Hervé<sup>a</sup>, Sandrine Perol<sup>b</sup>, Valérie Bernard<sup>c</sup>, Daphné Karila<sup>d</sup>, Eva Jali<sup>e</sup>, Aude Brac de la Perrière<sup>f</sup>, Virginie Grouthier<sup>g</sup>, Sophie Jonard-Catteau<sup>h</sup>, Philippe Touraine<sup>i</sup>, Corinne Fouveaut<sup>a</sup>, Geneviève Plu-Bureau<sup>b</sup>, Jean Michel Dupont<sup>a</sup>, Anne Bachelot<sup>i</sup>, Sophie Christin-Maitre<sup>d</sup>, Thierry Bienvenu<sup>a,w</sup>

<figure><img src="image_2.png" /></figure>

ARTICLE INFO

Edited by Wassim Y. Almawi

Keywords:

GDF9

Premature ovarian insufficiency

Penetrance

A B S T R A C T

**Background:** Primary ovarian insufficiency (POI) affects around 2–4% of women before the age of 40. Genetic factors play an important role in POI. The *GDF9* gene has been identified as a significant genetic contributor of POI. However, the pathogenicity and penetrance of *GDF9* variants remain uncertain.

**Methods:** A next-generation sequencing approach was employed to investigate the entire coding region of the *GDF9* gene in a cohort of 1281 patients with POI or diminished ovarian reserve (DOR). The frequency of each identified *GDF9* variant was then compared with that of the general population, taking into account the ethnicity of each individual.

**Results:** By screening the entire coding region of the *GDF9* gene, we identified 19 different variants, including 1 pathogenic frameshift variant. In total, 36 patients with POI/DOR (2.8%) carried at least one *GDF9* variant. With regard to missense variants, no significant overrepresentation of the most common variants was observed in our POI/DOR cohort in comparison to the general or specific ethnic subgroups. Only one homozygous subject had a frameshift loss of function variant.

**Conclusion:** This epidemiological study suggests that the vast majority of heterozygous missense variants could be considered as variants of uncertain significance and the homozygous loss-of-function variant could be considered as a pathogenic variant. The identification of a novel case of a homozygous POI patient with a heterozygous mother carrying the same variant with normal ovarian function strongly suggests that GDF9 syndrome is an autosomal recessive disorder.

**Abbreviations:** ACMG, American College of Medical Genetics and Genomics; DNA, Deoxyribonucleic acid; DOR, Diminished ovarian reserve; GATK, Genome analysis toolkit; GDF9, Growth Differentiation Factor 9; gnomAD, Genome Aggregation Database; FMR1, Fragile X Messenger Ribonucleoprotein 1; FSH, Follicle stimulating hormone; PCR, Polymerase chain reaction; POI, Primary ovarian insufficiency; VUS, Variants of unknown significance.

* Correspondent author at: Service de Médecine Génomique des Maladies de Système et d'Organe, Hôpital Cochin, 123 boulevard de Port-Royal, 75014 Paris, France.

E-mail address: thierry.bienvenu@inserm.fr (T. Bienvenu).

¹ These authors are co-first authors.

<footnote><sup>a</sup> Service de Médecine Génomique des Maladies de Système et d'Organe, Hôpital Cochin, APHP.Centre Université de Paris Cité, 75014 Paris, France</footnote>

<footnote><sup>b</sup> Unité de gynécologie médicale, APHP. Centre Université Paris Cité, Hôpital Cochin Port-Royal, 75014 Paris, France</footnote>

<footnote><sup>c</sup> Service de Chirurgie gynécologique et Médecine de la reproduction · Gynécologie médicale, CHU Bordeaux, Bordeaux, France</footnote>

<footnote><sup>d</sup> Service d'endocrinologie, diabétologie et médecine de la reproduction, APHP. Sorbonne Université, Hôpital Saint-Antoine, 75012 Paris, France</footnote>

<footnote><sup>e</sup> Service d'Endocrinologie, Hôpital de la Cavale Blanc, 29200 Brest, France</footnote>

<footnote><sup>f</sup> Service d'Endocrinologie, de diabétologie et des maladies métaboliques A, Hospices Civiles de Lyon, 69000 Lyon, France</footnote>

<footnote><sup>g</sup> Service d'Endocrinologie, Diabétologie et Nutrition, Hôpital Haut-Lévèque, CHU de Bordeaux, 33000 Bordeaux, France</footnote>

<footnote><sup>h</sup> Département d'assistance médicale à la procréation, Hôpital Jeanne de Flandre, 59000 Lille, France</footnote>

<footnote><sup>i</sup> Département d'Endocrinologie et médecine de la reproduction, APHP. Sorbonne Université, Pitié-Salpetrière Hospital, Center for Rare Endocrine and Gynecological Disorders, ERN-HCP, Paris, France</footnote>
# 1. Introduction

Premature ovarian insufficiency (POI) occurs in 2–4 % of women. Various factors can lead to the ovarian follicular depletion or dysfunction, including iatrogenic factors, environmental factors, autoimmune diseases, and genetic defects (abnormal karyotype, X-fragile and *FMRI* premutation). Considering that 10–30 % of POI cases have familial inheritance and/or genetic causes (Qin et al., 2015), more than one hundred of genes have been implicated in the aetiology of POI. These genes are involved in various key biological processes in the ovary, such as meiosis, and DNA damage repair, homologous recombination, follicle development, granulosa cell differentiation and proliferation, and ovulation. Although there is no specific consensus or international recommendations, next-generation sequencing using panel-specific genes, whole exome sequencing or whole genome sequencing is revolutionising the field of POI and identifying a high number of genetic variants. These approaches can be very highly beneficial in elucidating the aetiology of POI. However, a significant proportion of variants are often rare and their role in the pathogenesis is uncertain, and are therefore classified as variants of unknown significance (VUS). Furthermore, incomplete penetrance and variable clinical expressivity limit their clinical interest and utility in genetic counselling.

Among the genes involved in ovarian development and folliculogenesis, Growth Differenciation Factor 9 (*GDF9*) was one of the first to be described in human POI (Takebayashi et al., 2000). Between 2005 and 2007, five independent studies identified heterozygous *GDF9* variants in different populations of women with POI (Dixit et al., 2005; Laisse et al., 2006; Chand et al., 2006; Kovanci et al., 2007; Zhao et al., 2007). The pathogenicity of these variants was suggested by their frequency in the population database, familial segregation (or autosomal dominant inheritance from the father), and effect according to in silico analysis. Moreover, a few functional studies have been performed showing that transfection of several *GDF9* missense mutants reduces or abolishes the production of mature proteins and reduces the biological activities of conditioned media from human embryonic kidney 293F cells compared to the corresponding wild-type *GDF9* (Inagaki and Shimasaki, 2010; Wang et al., 2013; Simpson et al., 2014).

However, the pathogenesis of several *GDF9* variants and the context of autosomal dominant inheritance are questionable for at least four independent reasons: 1- some variants do not appear to be very rare, with a prevalence of 1 %-4% in certain ethnic groups; 2- *GDF9* missense and frameshift variants have been identified in twin mothers (Palmer et al., 2006) and in polycystic ovarian syndrome in the absence of POI (Takebayashi et al., 2000); 3- a homozygous loss-of-function variant (c.783del; p.Ser262HisfsTer2) was identified in a 19-year-old woman with POI (França et al., 2018), while her sister and her mother carrying the variant were unaffected, suggesting an autosomal recessive inheritance; and 4- mice carrying a null *Gdf9* mutation had POI, while the heterozygous female did not show an abnormal phenotype (Carabatsos et al., 1998).

All these recent data raised questions about the pathogenicity of *GDF9* variants in the context of autosomal dominant inheritance. To analyse the pathogenicity of the different variants according to specific ethnic origins, we compared the prevalence of *GDF9* variants identified in our large cohort of 1281 unrelated patients with POI/DOR from different ethnic-geographical origins with the frequency reported in the general population in the gnomAD database.

# 2. Materials & methods

## 2.1. Patients

We recruited 1281 unrelated women under 40 years of age over a 3-year period (January 2020 to July 2023), 704 with idiopathic, sporadic or familial POI, primary or secondary amenorrhoea, elevated FSH (>25 UI/L) on two measurements, and 577 with DOR (diminished ovarian

reserve) defined by a slightly elevated FSH level and a reduced number of ovarian follicles. We excluded from the statistical analysis 65 POI patients with a known aetiology of POI (such as medical treatments (radiotherapy, chemotherapy, pelvic surgery), autoimmune diseases (autoimmune thyroiditis, systemic lupus erythematosus), genetic pathogenic variants, primary adrenal insufficiency, and Perrault syndrome). The women were divided into four groups according to their ethnic origin: European (n = 739), North African (n = 160), Sub-Saharan African (n = 306) and Asian (n = 11). To compare the frequencies of the variants, we used the public gnomADv3 and gnomADv4 databases (https://gnomad.broadinstitute.org/)(v2.1.2). Our work complies with the principles laid down in the Declaration of Helsinki. All POI/DOR patients gave informed written consent to participate and signed an informed consent form approved by the local ethics committee.

## 2.2. Next generation Sequencing, mutation validation and in silico analysis

Genomic DNA was extracted from peripheral blood samples of women with POI/DOR and purified using the Chemagic 360 Nucleic Acid Extractor (Perkin Elmer) and the Wizard Genomic DNA Promega Kit (Promega Corporation). A custom target capture array, previously described (Jordan et al., 2024), was designed by Roche NimbleGen to cover seven selected known causative genes, including *GDF9* (NM_005260.7) (Qin et al., 2015; Luo et al., 2023). Briefly, 1 µg of genomic DNA was fragmented using an enzymatic approach. The sample library was prepared using a KAPA Library Preparation Kit (KAPA, KR0935). Target regions were captured using the SeqCap EZ library Kit (Roche NimbleGen), followed by multiplex sequencing on the Illumina MiSeq System at an average coverage of 200x according to the manufacturer's recommendations. Following the filtration of substandard reads, the human genome reference (NCBI build37/hg19 version) was utilized in mapping the remaining reads through the Burrows-Wheeler Aligner (BWA). The genome analysis toolkit (GATK) and Polyweb (http://www.polyweb.fr/) were applied for variant identification and filtration as previously described (https://www.broadinstitute.org/gatk/guide/topic?name=bestpractices) (Jordan et al., 2024). All calls with a read coverage ≤ 5×, a Phred scaled SNP quality of ≤ 20 and a MAF > 3 % were filtered out. Variants were annotated using an in-house Paris Descartes bioinformatics platform pipeline based on the Ensembl database (release 67). Variant classification followed the latest ACMG guidelines. All rare genetic variants were verified by Sanger sequencing on an ABI3500 xL DX DNA analyser according to the manufacturer's instructions (Applied Biosystems, Life Technologies). Primer sequences and positions, PCR conditions and product sizes are available on request. The pathogenic effects of each variant were assessed using various bioinformatics tools (CADD (combined annotation dependent depletion); Alamut v.2.4 (Interactive Biosoftware) (https://www.interactive-biosoftware.com/alamut)).

## 2.3. Statistical analysis

Statistical analyses were performed using SAS software (SAS institute, Inc.n Cary, NC) and the R environment (https://www.R-project.org/.) Fisher's exact test was used to assess the differences between the cohort and the general population and to examine the estimated odds ratio. Data are presented as a percentage or mean and SD for quantitative variables. P < 0.05 was considered as statistically significant.

# 3. Result

Screening of the entire coding region of the *GDF9* gene allowed us to identify 19 different variants, 1 frameshift variant, and 18 different missense variants (Fig. 1; Table 1). Eleven variants were located in the propeptide region (amino acids 25–319) and 8 in the mature chain (aa
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Location of the different GDF9 variants identified in the cohort of women with POI/DOR (n = 1281).</figcaption></figure>

320–454). One variant p.(Ser325Cys) was located in the phosphorylation site of the protein.

Only one of these variants was not previously reported (gnomADv4; Table 1). In total, 36 patients with POI/DOR (2.8%) carried out at least one GDF9 variant. Thirty-five patients carried out a heterozygous variant and one individual was homozygous for a frameshift variant. None of these patients had another pathogenic variant in the other genes tested.

Although several variants (e.g. p.(Lys67Gln), p.(Pro103Ser), p. (Ser425Arg), and p.(Arg454Cys)), identified in our study, have shown different degrees of in vitro functional impairment in vitro, including defects in transcriptional activity and biological activities (Inagaki and Shimasaki, 2010; Wang et al., 2013), the pathogenicity of these variants remains unclear. Using data from our POI cohort, we compared the frequency of the GDF9 variants in our population of POI/DOR patients (n = 1216) and in the general population. When we compared the frequency of the variants in our POI/DOR population with the frequency in the general population considering only women (Table 1), no significant differences were observed for 11 rare variants (Fig. 2; for 14 variants using gnomADv3, Fig.S1). Overall, only eight variants were found to be significantly overrepresented in POI/DOR (Fig. 2). The odds ratio (OR) of the variant carriers ranged from 14.85 to infinity (Fig. 2). The clinical characteristics of POI patients carrying these variants are shown in Table 2.

It is worth noting that several GDF9 variants have been identified globally, though with a somewhat uneven geographical and ethnic distribution. It would appear that the variants p.(Arg58Gly), p. (Pro119Leu), p.(Met233Val) p.(Val402Ile), and p.(Pro417Leu) were most frequently reported in African/African Americans, whereas the variant p.(Lys67Gln) was predominantly reported in subjects of South Asian origin (Table 1). It would also appear that p.(Ser425Arg) is more commonly found in subjects of East Asian origin, while p.(Pro103Ser), p. (Thr121Ile), p.(Asn292Tyr), p.(Ser363Asn), p.(Arg393Trp), and c.1294del are more frequently observed in subjects of European origin (Table 1). Given these differences in distribution, we decided to perform an analysis of GDF9 variants with ethnic subgroups. When looking at the ethnic subgroups, no significant difference was observed for ten variants, which are mainly observed in certain ethnic populations (Fig.S2). However, six variants, including p.(Ala306Pro), p.(Ser325Cys), p. (Arg393Trp) and c.1294del remain overrepresented in POI/DOR when considering the general ethnic population (Fig.S1). In addition, bioinformatic prediction by both SIFT, Polyphen-2, CADD and REVEL

suggested no pathogenic effect for four variants, but a pathogenic effect for four variants (Table 1). Overall, two of these 18 missense variants (p. (Thr121Ile) and p.(Arg454Cys)) were considered likely to be benign, three were considered likely to be pathogenic ((p.(Ala306Pro), p. (Ser325Cys) and p.(Arg393Trp)) and the others were considered variants of uncertain significance (Table 1).

In contrast to the most previously described heterozygous missense variants, we report a homozygous 1-bp deletion (c.1294del) in the GDF9 gene in a French patient with primary-secondary amenorrhoea. At first presentation, a 23-year-old woman was characterised by elevated FSH (51 UI/L) and LH (luteinising hormone) (18 UI/L). The patient's height was 155 cm. Breast development and pubic hair were Tanner 5 and 5 (S5P5). The karyotype was 46,XX, determined by the analysis of at least 50 metaphases. IVF by oocyte donation was performed and she was pregnant but miscarried. She has one brother and 3 asymptomatic sisters. One of the 3 sisters has an asymptomatic child. The variant c.1294del followed the guideline of ACMG guideline to be considered pathogenic: it was very rare in all international databases; it was a null variant in a gene where loss-of-function is a known mechanism of disease. Both parents were probably heterozygous and the patient's mother went through the menopause at the age of 53.

## 4. Discussion

GDF9 variants are often described as one of the top 20 common genetic explanations for POI (Jiao et al., 2021; Luo et al., 2023). However, frameshift, nonsense and splice site variants are very rare in the general population and in POI cohorts. To date, only one frameshift variant and one large duplication variant have been identified in women with POI (Norling et al., 2014; França et al., 2018). In this study, we identified 1 rare loss-of-function variant (Table 1). However, the vast majority of GDF9 variants were missense variants for which pathogenicity remains elusive, especially as few missense variants are commonly observed in the general population. To help classify these variants, we analysed the OR values according to their molecular consequences and to their ethnic frequency.

In our large cohort of POI/DOR, we identified 18 different missense variants, and found no significant overrepresentation of 11 variants in our 1216 POI/DOR patients compared to the general population using data from the gnomADvs4 database. Overall, we observed a significant overrepresentation of only 7 missense variants in our POI/DOR cohort. The OR of these variant carriers varied between 14.85 and infinity
<caption>Table 1</caption>

GDF9 variants identified in the cohort of 1281 individuals. Results of pathogenicity prediction using different software (CADD, SIFT, Polyphen-2, Revel) are given for each variant. The results of the frequency in the gnomADv3 and V4 databases are given. Classification of variants according to the ACMG rules is shown. The position of each variant in the subunits of the preproprotein is indicated: the peptide signal (amino acids (aa) 1–24); the prodomain (aa 25–319); the mature region (aa 320–454). Nd: not identified; *, phosphorylation site.

<table><thead><tr><th>Sequence variation</th><th>Amino acid variation</th><th>ACMG criteria v3</th><th>ACMG classification v3</th><th>ACMG criteria v4</th><th>ACMG classification v4</th><th>Number of cases</th><th>gnomAD v3 (women)</th><th>Asian</th><th>European</th><th>African</th><th>Latino/ Admixed American</th><th>gnomAD v4 (women)</th><th>Asian</th><th>European</th><th>African</th><th>Latino/ Admixed American</th><th>CADD phred</th><th>SIFT</th><th>Polyphen-2</th><th>Revel</th></tr></thead><tbody><tr><td>c.109A > G p.(Ser37Gly)</td><td>PM2, BP4</td><td>Variant of Unknown Significance</td><td>PM2, BP4</td><td>Variant of Unknown Significance</td><td>1</td><td>5/77828</td><td>2/ 3662</td><td>1/39354</td><td>nd</td><td>2/6788</td><td>47/812320</td><td>5/ 23186</td><td>31/ 611572</td><td>nd</td><td>11/32462</td><td>4.25</td><td>Tolerated</td><td>Benign</td><td>Benign</td></tr><tr><td>c.172A > G p.(Arg58Gly)</td><td>PM2, BP4</td><td>Variant of Unknown Significance</td><td>PS4, PM2, BP4</td><td>Variant of Unknown Significance</td><td>2</td><td>10/77854</td><td>nd</td><td>nd</td><td>6/ 22146</td><td>4/6792</td><td>29/812480</td><td>nd</td><td>nd</td><td>20/ 41488</td><td>5/32486</td><td>8.30</td><td>Tolerated</td><td>Benign</td><td>Benign</td></tr><tr><td>c.199A > C p.(Lys67Gln)</td><td>PM2</td><td>Variant of Unknown Significance</td><td>BS2, PM2</td><td>Variant of Unknown Significance</td><td>1</td><td>0/77820</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>39/812456</td><td>36/ 23192</td><td>nd</td><td>nd</td><td>nd</td><td>23.00</td><td>Damaging</td><td>Possibly damaging</td><td>Uncertain</td></tr><tr><td>c.307C > T p.(Pro103Ser)</td><td>PM1, PP3, BS1</td><td>Variant of Unknown Significance</td><td>PM1, PP3, BS1, BS2</td><td>Variant of Unknown Significance</td><td>8</td><td>170/77842</td><td>nd</td><td>122/ 39356</td><td>6/ 22152</td><td>5/8492</td><td>2612/ 812450</td><td>2/ 23194</td><td>2115/ 611614</td><td>17/ 41494</td><td>22/32478</td><td>25.60</td><td>Tolerated</td><td>Probably damaging</td><td>Uncertain</td></tr><tr><td>c.356C > T p.(Pro119Leu)</td><td>PM1, PM2, BS1</td><td>Variant of Unknown Significance</td><td>PS4, PM1, PM2, BP4</td><td>Variant of Unknown Significance</td><td>2</td><td>28/77860</td><td>nd</td><td>nd</td><td>28/ 22148</td><td>nd</td><td>45/812504</td><td>nd</td><td>1/611658</td><td>42/ 41474</td><td>nd</td><td>26.60</td><td>Damaging</td><td>Probably damaging</td><td>Uncertain</td></tr><tr><td>c.362C > T p.(Thr121Ile)</td><td>BS1, BP4</td><td>Likely benign</td><td>BS1, BS2, BP4</td><td>Likely benign</td><td>1</td><td>39/77850</td><td>nd</td><td>20/39356</td><td>3/ 19304</td><td>nd</td><td>284/ 812478</td><td>nd</td><td>258/ 611632</td><td>7/ 41490</td><td>4/32486</td><td>15.71</td><td>Tolerated</td><td>Benign</td><td>Benign</td></tr><tr><td>c.364C > T p.(Arg122Trp)</td><td>PM2, BP4</td><td>Variant of Unknown Significance</td><td>PS4, PM2, BP4</td><td>Variant of Unknown Significance</td><td>1</td><td>5/77826</td><td>nd</td><td>1/39352</td><td>4/ 22138</td><td>nd</td><td>14/812462</td><td>nd</td><td>7/611638</td><td>6/ 41464</td><td>nd</td><td>23.90</td><td>Damaging</td><td>Possibly damaging</td><td>Benign</td></tr><tr><td>c.697A > G p.(Met233Val)</td><td>PM2, BP4</td><td>Variant of Unknown Significance</td><td>PM2, BP4</td><td>Variant of Unknown Significance</td><td>1</td><td>16/77864</td><td>nd</td><td>nd</td><td>16/ 22158</td><td>nd</td><td>31/812122</td><td>nd</td><td>nd</td><td>27/ 41498</td><td>2/32488</td><td>8.21</td><td>Tolerated</td><td>Benign</td><td>Uncertain</td></tr><tr><td>c.874G > T p.(Asn292Tyr)</td><td>PM2, BP4</td><td>Variant of Unknown Significance</td><td>PS4, PM2, BP4</td><td>Variant of Unknown Significance</td><td>1</td><td>1/77844</td><td>nd</td><td>1/39364</td><td>nd</td><td>nd</td><td>11/812426</td><td>nd</td><td>11/ 611606</td><td>nd</td><td>nd</td><td>16.47</td><td>Damaging</td><td>Possibly damaging</td><td>Uncertain</td></tr><tr><td>c.916G > C p.(Ala306Pro)</td><td>PM1, PM2, BP4</td><td>Variant of Unknown Significance</td><td>PS4, PM1, PM2, BP4</td><td>Likely pathogenic</td><td>1</td><td>1/77850</td><td>nd</td><td>nd</td><td>nd</td><td>1/6792</td><td>9/812336</td><td>nd</td><td>3/611508</td><td>nd</td><td>1/32482</td><td>3.17</td><td>Tolerated</td><td>Benign</td><td>Benign</td></tr><tr><td>c.947G > A p.(Arg316His)</td><td>PM2, PP3</td><td>Variant of Unknown Significance</td><td>PM2, PP3</td><td>Variant of Unknown Significance</td><td>1</td><td>1/77848</td><td>nd</td><td>1/39364</td><td>nd</td><td>nd</td><td>36/812286</td><td>14/ 23204</td><td>18/ 611468</td><td>nd</td><td>nd</td><td>23.40</td><td>Damaging</td><td>Probably damaging</td><td>Uncertain</td></tr><tr><td>c.973A > T p.(Ser325Cys)</td><td>PM1, PM2, PP3</td><td>Variant of Unknown Significance</td><td>PS4, PM1, PM2, PP3</td><td>Likely pathogenic</td><td>1</td><td>0/77842</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>0/809738</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>13.91</td><td>Damaging</td><td>Probably damaging</td><td>Uncertain</td></tr><tr><td>c.1088G > A p.(Ser363Asn)</td><td>PM1, PP3</td><td>Variant of Unknown Significance</td><td>PM1, PP3, BS2</td><td>Variant of Unknown Significance</td><td>1</td><td>23/77864</td><td>nd</td><td>19/39364</td><td>nd</td><td>1/6790</td><td>388/ 812320</td><td>14/ 23194</td><td>338/ 611482</td><td>1/ 41492</td><td>6/32486</td><td>22.50</td><td>Damaging</td><td>Possibly damaging</td><td>Damaging</td></tr><tr><td>c.1177C > T p.(Arg393Trp)</td><td>PM1, PM2, PP3</td><td>Variant of Unknown Significance</td><td>PS4, PM1, PM2, PP3</td><td>Likely pathogenic</td><td>1</td><td>0/77830</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>7/810254</td><td>nd</td><td>6/609652</td><td>nd</td><td>1/32468</td><td>26.10</td><td>Damaging</td><td>Possibly damaging</td><td>Damaging</td></tr><tr><td>c.1204G > A p.(Val402Ile)</td><td>PM1, PP3, BS1</td><td>Variant of Unknown Significance</td><td>PM1, PP3, BS1</td><td>Variant of Unknown Significance</td><td>1</td><td>103/77832</td><td>nd</td><td>1/39352</td><td>98/ 22138</td><td>nd</td><td>218/ 811394</td><td>nd</td><td>1/610656</td><td>200/ 41460</td><td>8/32486</td><td>24.30</td><td>Damaging</td><td>Possibly damaging</td><td>Uncertain</td></tr><tr><td>c.1250C > T p.(Pro417Leu)</td><td>PM1, PP3, BS1</td><td>Variant of Unknown Significance</td><td>PM1, PP3, BS1, BS2</td><td>Variant of Unknown Significance</td><td>1</td><td>164/77840</td><td>nd</td><td>1/39354</td><td>157/ 22150</td><td>4/6788</td><td>382/ 811626</td><td>4/ 23172</td><td>30/ 610874</td><td>325/ 41466</td><td>8/32482</td><td>27.90</td><td>Damaging</td><td>Possibly damaging</td><td>Damaging</td></tr><tr><td>c.1275C > A p.(Ser425Arg)</td><td>PM1, PM2, BS1</td><td>Variant of Unknown Significance</td><td>PM1, PM2, BS2</td><td>Variant of Unknown Significance</td><td>1</td><td>11/77842</td><td>nd</td><td>4/ 22156</td><td>nd</td><td>nd</td><td>66/809738</td><td>46/ 22960</td><td>1/609152</td><td>11/ 41418</td><td>nd</td><td>23.60</td><td>Tolerated</td><td>Possibly damaging</td><td>Damaging</td></tr><tr><td>c.1294del p.(Ile432LeufsTer15)</td><td>PVS1, PS4, PM2</td><td>Likely pathogenic</td><td>PVS1, PS4, PM2</td><td>Likely pathogenic</td><td>1</td><td>0/77850</td><td>nd</td><td>nd</td><td>nd</td><td>nd</td><td>7/810576</td><td>nd</td><td>6/609896</td><td>nd</td><td>1/32480</td><td>28.00</td><td></td><td></td><td></td></tr><tr><td>c.1360C > T p.(Arg454Cys)</td><td>PP3, BS1, BP6</td><td>Likely benign</td><td>PP3, BS1, BP6</td><td>Likely benign</td><td>9</td><td>242/77824</td><td>3/ 1166</td><td>166/ 39352</td><td>14/ 22142</td><td>nd</td><td>2974/ 812008</td><td>65/ 23136</td><td>2533/ 611258</td><td>30/ 41478</td><td>126/32480</td><td>32.00</td><td>Damaging</td><td>Possibly damaging</td><td>Damaging</td></tr></tbody></table>
<figure><img src="image_4.png" /><figcaption>Fig. 2. Forest plot showing the odds ratios for the risk of POI/DOR in women with POI/DOR associated with GDF9 variants (POI/DOR cohort versus controls from the gnomADv4 general population controls). Note: Confidence interval (min = minimum, max = maximum) is shown.</figcaption></figure>

<table><caption>**Table 2**

Clinical characteristics of POI/DOR patients with potentially pathogenic GDF9 variants. Nd = not determined. G/P (Gravida Para Scoring, G: pregnancy; P: live birth).</caption><thead><tr><th>Patient ID</th><th>18ME00603</th><th>2121ME000053</th><th>2120ME000141</th><th>2120ME000224</th></tr></thead><tbody><tr><td>Variant</td><td>c.973A > T</td><td>c.1294del</td><td>c.1177C > T</td><td>c.916G > C</td></tr><tr><td>Aminoacid substitution</td><td>p.(Ser325Cys)</td><td>p.(Ile432Leufs15)</td><td>p.(Arg393Trp)</td><td>p.(Ala306Pro)</td></tr><tr><td>POI/DOR</td><td>POI</td><td>POI</td><td>POI</td><td>POI</td></tr><tr><td>Amenorrhea (P/S)</td><td>S</td><td>Primary-secondary</td><td>S</td><td>S</td></tr><tr><td>Ethnic origin</td><td>South-American (Peru)</td><td>European (Portugal)</td><td>African (Ivory Coast)</td><td>European</td></tr><tr><td>Current age (year)</td><td>37</td><td>41</td><td>34</td><td>45</td></tr><tr><td>Age of first menstruation (years)</td><td>15</td><td>12</td><td>14</td><td>13</td></tr><tr><td>Age of amenorrhea (years)</td><td>34</td><td>12</td><td>32</td><td>30</td></tr><tr><td>No of pregnancies (G/P)</td><td>G0P0</td><td>G1P0 (egg donation)</td><td>G3P2</td><td>G3P3</td></tr><tr><td>FSH (IU/L)</td><td>70</td><td>51</td><td>137</td><td>25</td></tr><tr><td>LH (IU/L)</td><td>35</td><td>18</td><td>75.5</td><td>9</td></tr><tr><td>Estradiol (pg/ml)</td><td>57</td><td>nd</td><td>41</td><td>51</td></tr><tr><td>AMH (ng/ml)</td><td>0.02</td><td>nd</td><td>0.014</td><td>nd</td></tr><tr><td>BMI (kg/m<sup>2</sup>)</td><td>24.5</td><td>nd</td><td>30</td><td>nd</td></tr><tr><td>Height (cm)</td><td>155</td><td>nd</td><td>158</td><td>nd</td></tr><tr><td>Syndromic features</td><td>none</td><td>none</td><td>none</td><td>none</td></tr><tr><td>Associate phenotype</td><td>none</td><td>none</td><td>none</td><td>none</td></tr></tbody></table>

(Fig. 2). Furthermore, when we considered the ethnic subgroups, we found no significant difference for two of these variants (p.(Arg58Gly); p.(Pro119Leu)), suggesting that there are ethnic-specific variants (Fig. S2). In addition, bioinformatic predictions suggested that only one overrepresented missense variant (p.Arg393Trp) was likely to be deleterious. Overall, our epidemiological study using the ACMG classification showed that of the 18 rare GDF9 missense variants identified, two are likely to be benign, three are likely to be pathogenic, and the others were considered variants of uncertain significance.

Until recently, the vast majority of human GDF9 variants described so far in different POI cohorts were heterozygous missense variants. A

mutational screening of GDF9 in 127 patients with POI revealed two rare heterozygous missense variants in six patients with secondary amenorrhea of the cohort (Dixit et al., 2005). In 2006, Laissue and colleagues identified the heterozygous p.(Ser186Tyr) variant in a patient with secondary amenorrhoea from a European cohort of 203 women with POI (Laissue et al., 2006). The GDF9 variant was also found as a rare explanation for POI in the US cohort. In 61 subjects, most of them Caucasian, only one patient carried a heterozygous missense variant (p. (Pro103Ser)) (Kovanci et al., 2007). All these previous data suggest that POI due to GDF9 variant is a dominant autosomal disorder.

In contrast to most previously described heterozygous missense
mutations, we report a homozygous 1-bp deletion (c.1294del) in the *GDF9* gene in a French patient with primary-secondary amenorrhoea. This was a null variant in a gene where loss-of-function is a known disease mechanism. According to the Prosite_SIB protein domain database, the TGF-β domain region in GDF9 is located between 316 and 454 amino acids. In addition, variants in the mature region could affect the normal growth and maturation of ovarian cells. Our variant (p.Ile432-LeufsTer15) could result in a truncated protein lacking the end of the TGF-β domain (aa 432–454), leading to a loss-of-function of the GDF9 protein. Although maternal DNA was not available, our next-generation sequencing analysis excluded the presence of a deletion affecting one or more exons of the *GDF9* gene, suggesting that the variant identified in the POI patient is a homozygous deletion of A at position c.1294. As the patient's mother carrying the variant was unaffected (menopause at the age of 53), our report supports the recent conclusion by Franca and colleagues that *GDF9* is involved in a autosomal recessive disorder (França et al., 2018). These conclusions are supported by Gdf9-null mouse models. Gdf9-null female mice are infertile due to blockage at the primary follicle stage, whereas heterozygous Gdf9<sup>+/-</sup> females are fertile (Carabatsos et al., 1998).

However, it should be noted that our study has several limitations. Firstly, there is significant phenotypic heterogeneity within the group of women with fertility disorders (POI/DOR). It is possible that some of these women may have had at least one child before POI or after POI, with or without the use of assisted reproduction. Secondly, the gno-mADv4 database brings together data from over 807,162 individuals through a worldwide collaborative data sharing effort. The majority of the sequence data were generated for case-control studies of common adult diseases and may include women with known or unknown POI (~2% in the 35–40-year age group), who are considered to be healthy. Finally, it is possible that another pathogenic variant exists in different genes, which would suggest that POI has a polygenic or oligogenic origin (Shekari et al., 2023). Whole genome sequencing should be considered in the future management of these patients, especially in familial cases of POI or in the presence of a heterozygous pathogenic GDF9 variant.

Despite these limitations and unresolved issues, our analysis identified one rare loss-of-function variant, three likely pathogenic missense variants (including one novel one), two likely benign missense variants and 13 missense variants of uncertain significance. Our report strongly supports the notion that GDF9 syndrome is an autosomal recessive disorder. However, we cannot exclude that biallelic variants show more severe phenotype than those with heterozygous variants, and that the heterozygous variants are causally associated with pathogenic variants in other genes. Although GDF9 variants explain less than 0.1 % of POI/DOR, these data support routine genetic screening of patients in clinical practice.

### CRediT authorship contribution statement

**Pénélope Jordan:** Writing – review & editing, Methodology, Investigation, Formal analysis, Data curation. **Camille Verebi:** Writing – review & editing, Writing – original draft, Validation, Software, Methodology, Data curation. **Bérénice Hervé:** Sandrine Perol: Writing – review & editing, Resources, Investigation, Formal analysis, Data curation. **Valérie Bernard:** Writing – review & editing, Resources, Methodology, Investigation, Formal analysis, Data curation. **Daphné Karila:** Writing – review & editing, Resources, Methodology, Investigation, Formal analysis, Data curation. **Eva Jali:** Resources, Methodology, Investigation, Formal analysis, Data curation. **Aude Brac de la Perriere:** Writing – review & editing, Resources, Methodology, Investigation, Formal analysis, Data curation. **Virginie Grouthier:** Resources, Methodology, Formal analysis, Data curation. **Sophie Jonard-Catteau:** Writing – review & editing, Writing – original draft, Validation, Resources, Methodology, Investigation, Data curation, Conceptualization. **Philippe Touraine:** Writing – review & editing, Resources, Investigation, Formal analysis. **Corinne Fouveaut:** Methodology,

Formal analysis, Data curation. **Geneviève Plu-Bureau:** Resources, Methodology, Formal analysis, Data curation. **Jean Michel Dupont:** Writing – review & editing, Validation, Supervision, Resources, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Anne Bachelot:** Writing – review & editing, Resources, Investigation, Formal analysis, Data curation. **Sophie Christin-Maitre:** Writing – review & editing, Writing – original draft, Validation, Resources, Methodology, Investigation, Data curation, Conceptualization. **Thierry Bienvenu:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Resources, Methodology, Investigation, Formal analysis, Data curation, Conceptualization.

### Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Bienvenu reports was provided by Public Assistance Hospitals Paris. Bienvenu reports a relationship with Public Assistance Hospitals Paris that includes: employment and non-financial support. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Data availability

The data that support the findings of the study are available from the corresponding author and the first author upon reasonable request.

### Acknowledgement

The authors would like to sincerely acknowledge all clinicians for their continuous support in providing the blood samples and clinical information of the patients. We extend our thanks to all the study participants for their valuable involvement to execute this study successfully.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.gene.2024.148734.

### References

* Carabatsos, M.J., Elvin, J., Matzuk, M.M., Albertini, D.F., 1998. Characterization of oocyte and follicle development in growth differentiation factor-9-deficient mice. Dev. Biol. 204 (2), 373-384. https://doi.org/10.1006/dbio.1998.9087. PMID: 9882477.

* Chand, A.L., Ponnampalam, A.P., Harris, S.E., Winship, I.M., Shelling, A.N., 2006. Mutational analysis of BMP15 and GDF9 as candidate genes for premature ovarian failure. Fertil. Steril. 86 (4), 1009-1012. https://doi.org/10.1016/j.fertnstert.2006.02.107. PMID: 17027369.

* Dixit, H., Rao, L.K., Padmalatha, V., Kanakavalli, M., Deenadayal, M., Gupta, N., et al., 2005. Mutational screening of the coding region of growth differentiation factor 9 gene in Indian women with ovarian failure. Menopause. 12 (6), 749-754. https://doi.org/10.1097/01.gme.0000184424.96437.7a. Epub 2005 Nov 8 PMID: 16278619.

* França, M.M., Funari, M.F.A., Nishi, M.Y., Narcizo, A.M., Domenice, S., Costa, E.M.F., et al., 2018. Identification of the first homozygous 1-bp deletion in GDF9 gene leading to primary ovarian insufficiency by using targeted massively parallel sequencing. Clin. Genet. 93 (2), 408-411. https://doi.org/10.1111/cge.13156. Epub 2017 Dec 26 PMID: 29044499.

* Inagaki, K., Shimasaki, S., 2010. Impaired production of BMP-15 and GDF-9 mature proteins derived from proproteins WITH mutations in the proregion. Mol. Cell. Endocrinol. 328 (1-2), 1-7. https://doi.org/10.1016/j.mce.2010.05.017. Epub 2010 Jun 12. PMID: 20547206; PMCID: PMC2934881.

* Jiao, S.Y., Yang, Y.H., Chen, S.R., 2021. Molecular genetics of infertility: loss-of-function mutations in humans and corresponding knockout/mutated mice. Hum. Reprod. Update. 27 (1), 154-189. https://doi.org/10.1093/humupd/dmaa034. PMID: 33118031.

* Jordan, P., Verebi, C., Perol, S., Grotto, S., Fouveaut, C., Christin-Maitre, S., et al., 2024. NOBOX gene variants in premature ovarian insufficiency: ethnicity-dependent insights. J. Assist. Reprod. Genet. 41 (1), 135-146. https://doi.org/10.1007/s10815-023-02981-y. Epub 2023 Nov 3. PMID: 37921973; PMCID: PMC10789696.
Kovanci, E., Rohozinski, J., Simpson, J.L., Heard, M.J., Bishop, C.E., Carson, S.A., 2007. Growth differentiating factor-9 mutations may be associated with premature ovarian failure. Fertil. Steril. 87 (1), 143-146. https://doi.org/10.1016/j.fertnstert.2006.05.079. Epub 2006 Dec 6 PMID: 17156781.

Laissue, P., Christin-Maitre, S., Touraine, P., Kutten, F., Ritvos, O., Aittomaki, K., et al., 2006. Mutations and sequence variants in GDF9 and BMP15 in patients with premature ovarian failure. Eur. J. Endocrinol. 154 (5), 739-744. https://doi.org/10.1530/eje.1.02135. PMID: 16645022.

Luo, W., Ke, H., Tang, S., Jiao, X., Li, Z., Zhao, S., et al., 2023. Next- generation sequencing of 500 POI patients identified novel responsible monogenic and oligogenic variants. J. Ovarian. Res. 16 (1), 39. https://doi.org/10.1186/s13048-023-01104-6. PMID: 36793102; PMCID: PMC9930292.

Norling, A., Hirschberg, A.L., Rodriguez-Wallberg, K.A., Iwarsson, E., Wedell, A., Barbaro, M., 2014. Identification of a duplication within the GDF9 gene and novel candidate genes for primary ovarian insufficiency (POI) by a customized high-resolution array comparative genomic hybridization platform. Hum. Reprod. 29 (8), 1818-1827. https://doi.org/10.1093/humrep/deu149. Epub 2014 Jun 17. PMID: 24939957; PMCID: PMC4093997.

Palmer, J.S., Zhao, Z.Z., Hoekstra, C., Hayward, N.K., Webb, P.M., Whiteman, D.C., et al., 2006. Novel variants in growth differentiation factor 9 in mothers of dizygotic twins. J. Clin. Endocrinol. Metab. 91 (11), 4713-4716. https://doi.org/10.1210/jc.2006-0970. Epub 2006 Sep 5 PMID: 16954162.

Qin, Y., Jiao, X., Simpson, J.L., Chen, Z.J., 2015. Genetics of primary ovarian insufficiency: new developments and opportunities. Hum. Reprod. Update. 21 (6),

787-808. https://doi.org/10.1093/humupd/dmv036. Epub 2015 Aug 4. PMID: 26243799; PMCID: PMC4594617.

Shekari, S., Stankovic, S., Gardner, E.J., Hawkes, G., Kentistou, K.A., Beaumont, R.N., et al., 2023. Penetrance of pathogenic genetic variants associated with premature ovarian insufficiency. Nat. Med. 29 (7), 1692-1699. https://doi.org/10.1038/s41591-023-02405-5. Epub 2023 Jun 22 PMID: 37349538.

Simpson, C.M., Robertson, D.M., Al-Musawi, S.L., Heath, D.A., McNatty, K.P., Ritter, L.J., et al., 2014. Aberrant GDF9 expression and activation are associated with common human ovarian disorders. J. Clin. Endocrinol. Metab. 99 (4), E615-E624. https://doi.org/10.1210/jc.2013-3949. Epub 2014 Jan 17 PMID: 24438375.

Takebayashi, K., Takakura, K., Wang, H., Kimura, F., Kasahara, K., Noda, Y., 2000. Mutation analysis of the growth differentiation factor-9 and -9B genes in patients with premature ovarian failure and polycystic ovary syndrome. Fertil. Steril. 74 (5), 976-979. https://doi.org/10.1016/s0015-0282(00)01539-9. PMID: 11056243.

Wang, T.T., Ke, Z.H., Song, Y., Chen, L.T., Chen, X.J., Feng, C., et al., 2013. Identification of a mutation in GDF9 as a novel cause of diminished ovarian reserve in young women. Hum. Reprod. 28 (9), 2473-2481. https://doi.org/10.1093/humrep/det291. Epub 2013 Jul 12 PMID: 23851219.

Zhao, H., Qin, Y., Kovanci, E., Simpson, J.L., Chen, Z.J., Rajkovic, A. 2007. Analyses of GDF9 mutation in 100 Chinese women with premature ovarian failure. Fertil. Steril. 88(5):1474-6. doi: 10.1016/j.fertnstert.2007.01.021. Epub 2007 May 7. PMID: 17482612; PMCID: PMC2767161.