We have used whole-genome array-CGH to identify microdeletions and microduplications in a cohort of 60 women presenting an OF. Search for genomic imbalances by microarray in patients with OF has become challenging and allowed in previous studies the identification of CNVs throughout the genome, on X chromosome and other regions. These rearrangements could affect known gonadal genes or candidate gonadal genes [
11,
14‐
17]. As indicated in the study of McGuire et al., these CNVs are frequently located on autosomes suggesting that candidate genes for OF are located on them [
14]. Aboura et al. have shown, in their study of 99 patients, 8 statistically different CNVs from control population, in 1p21.1, 5p14.3 (
DNAH5), 5q13.2 (
NAIP), 6p25.3 (
DUSP22), 14q32.33 (
AKT1), 16p11.2 (
NUPR1), 17q12, and Xq28, with five genes potentially involved in reproduction [
15]. Ledig et al. identified 44 significant CNVs in 74 POF and ovarian dysgenesis, with genes involved in meiosis as
PLCB1, RB1CC1, MAP4K4, genes involved in DNA repair as
RBBP8 and genes involved in folliculogenesis or male fertility as
IMMP2L, FER1L6, MEIG1 [
16]. McGuire et al. identified 24 autosomal CNVs in 89 patients, with genes linked to abnormal reproductive phenotypes in mouse models (
SYCE1 in 10q26.3 and
CPEB1 in 15q25.2) [
14]. Norling et al. have shown 11 significant CNVs in 26 POF, most of them involving new regions and candidate genes (e.g.
MCTP2 on 15q26.2,
CACNAIC on 12p13.33) [
17]. In the present study in 60 women presenting OF, nine candidate CNVs were likely to be involved in fertility. Anomalies of genes potentially involved in primordial follicles activation or folliculogenesis might explain the occurrence of this feature.
CNVs already linked to POF
A 3.6 Mb 8p23.2 duplication involving the
CSMD1 gene (
Cub and sushi multiple domains 1, OMIM 608397) was identified in patient 7. A 680 kb duplication involving this gene was observed in the study of McGuire et al. (patient POF-45) [
14]. Expression analyses have shown that CSMD1 can be expressed in follicles and corpus luteum of the female reproductive system [
18]. These data (recurrence of the variant and follicular expression) suggest that
CSMD1 could play a role in folliculogenesis. However,
CSMD1 has also been associated with autistic spectrum disorders and schizophrenia and is a known target of mir-137, a microRNA that regulates neuronal maturation and adult neurogenesis [
19]. Futhermore,
CSMD1, as tumor suppressor gene, has been associated with head and neck squamous cell carcinoma and with the tumorigenesis of several other epithelial cancers, including breast cancer with rare variants (deletions) predisposing individuals to breast cancer [
20].
In the study of McGuire et al. [
14], a 160 kb deletion at 10q26.3 (including
CYP2E1 and
SYCE1) was potentially linked to the POF phenotype. In our study, a 123 kb duplication of the same region was observed in patient 43 suggesting a role of this variant in gonadal development.
SYCE1 (
Synaptonemal complex central element 1, OMIM 611486) has been previously shown in animal models to be linked with the occurrence of POF. It is also involved in chromosome synapsis and recombination during meiosis. Recently, a homozygous mutation in
SYCE1 was identified by exome sequencing in two sisters with POF [
21].
CNVs involving other genes associated to cell division and chromosome segregation
Several genes encode proteins involved in cohesion, kinetochores and motor functions. It is supposed that CNVs of these genes could lead to errors in meiosis and gametogenesis. In patient 20, a 741 kb duplication at 2q14 involving the 5′end of
CLASP1 (
CLIP-associated protein 1, OMIM 605852) was detected. CLASP proteins are non-motile proteins associated with microtubules and interacting with CLIP proteins.
CLASP1 is required for the kinetochore-microtubule interaction and allows the mitotic spindle to exhibit normal dynamic behavior. It remains at the kinetochores upon the completion of chromosome congression and throughout anaphase, suggesting that it may modulate the dynamics of attached microtubules throughout mitosis [
22]. CLASP1-Astrin-Kif2b complex acts as a central switch at kinetochores that defines mitotic progression and promotes fidelity by temporally regulating kinetochore-microtubule attachments [
23]. Based on studies in the Xenopus meiotic spindle, Xorbit/CLASP also appears as a critical factor linking chromosome segregation to microtubules dynamics during meiosis [
24].
In patient 25, a 113 kb duplication at 13q34 involving
CDC16 (
Cell division cycle 16, OMIM 603461) was observed.
CDC16 is one of several subunits of the Anaphase-Promoting Complex (APC), which functions at the metaphase-to-anaphase transition of the cell cycle and is regulated by spindle checkpoint proteins. It is a core tetratricopeptide repeat (TPR)-containing protein stabilized by CDC26 in the APC and it is supposed that the stable complex CDC26-CDC16 may serve as a platform for assembling higher-order multi-TPR complex required for APC function [
25]. It has been showed that CDC16 is essential for cell division in human cells with a knockdown leading to a mitotic arrest [
26]. Defect in APC/C activity seems to be involved in the waves of oocyte degeneration occurring during shift from mitosis to meiosis, final stages of meiotic prophase I and at the breakdown of the oocyte nests preluding the primordial follicle assembly [
27]. Furthermore, CDC16 is part of TBC1D7 (Tre2-Bub2-Cdc16-1 domain family member 7), a subunit of the Tuberous Sclerosis Complex (TSC) in the PI3K/AKT/mTORC1 pathway [
28] for which some of the key components have implicated in spermatogenesis and oogenesis [
29].
In patient 21, a 559 kb duplication at 2p23.3 involving
CENP-A (
Centromere protein A, OMIM 117139) was observed. CENP-A is a histone H3-like protein involved in centromeric nucleosome formation. In mouse, Cenpa is essential for kinetochore targeting of Cenpc and plays an early role in organizing centromeric chromatin at interphase. The chromosomes seen in the null
Cenpa embryos appeared morphologically more condensed and scattered than those of normal embryos and no discernible mitotic chromosomes were apparent in the 6.5-day null embryos examined, suggesting cessation of mitosis at this point. The study suggests a critical epigenetic function for
CENP-A in marking a chromosomal region for centromere formation [
30].
CNVs involving genes associated with ciliary development and/or function
In patient 21, additionally to the
CENP-A duplication, a 21q22.3 223 kb deletion involving
RSPH1 (
Radial spoke head 1 homolog, OMIM 609314) was identified.
RSPH1 is involved in primary ciliar dyskinesia (PCD), a disorder with male infertility due to dysmotility of spermatozoa and reduced fertility or a history of ectopic pregnancies in affected women. PCD is a genetically heterogeneous, autosomal-recessive disorder with mutations reported in 28 genes (most frequently
DNAH5). Loss-of-function mutations of
RSPH1 have been associated to a milder clinical phenotype, as compared with patients with PCD with typical ultrastructural defects or mutations in genes that are commonly associated with PCD [
31]. It is interesting to note that patient 21 presents bronchiectasis and ciliary anomalies of the ducts which can be explained by loss-of-function of this gene and lead us to sequence the second allele of this gene.
In patient 29, a 185 kb duplication at 9p13.3 involving
UBAP1 (OMIM 609787),
KIF24 (OMIM 613747),
NUDT2 (OMIM 602852),
KIAA 1161 and
C9orf24 was observed. Among these genes,
KIF24 (
Kinesin family member 24, OMIM 613747) encodes a kinesin-13 motor protein that preferentially localizes to the distal end of mother centrioles. It possesses microtubule-depolymerizing activity and regulates cilia formation in cycling cells by depolymerizing centriole microtubules. In quiescent cells,
KIF24 is detected at the basal body of the primary cilium and its overexpression suppresses ciliogenesis [
32]. Thus
KIF24 has a role in the regulation of ciliogenesis which is important during embryonic development and in adult physiology [
33].
CNVs involving genes linked with known gonadal genes or with expression in female genital tract
In patient 47, a 88 kb duplication at 15q21.1 was identified. This CNV involve the 5′ end of
SEMA6D (
Semaphorin 6D, OMIM 609295), a gene potentially linked with
FOXO1. It has indeed been showed that two potential targets of
SEMA6D, the transcription factors
FOS and
FOXO1, were both increased in SEMA6D-high patients in case of breast cancer [
34]. A gain or loss of function of S
EMA6D could then result in a dysregulation in folliculogenesis as
FOXO1, expressed in human luteinized mural granulosa cells, is supposed to be involved in human folliculogenesis and luteinization [
35].
A 44 kb deletion at 1p13.31 involving
KIAA1324 (
Estrogen-induced gene 121 or EIG121, OMIM 611298) was identified in patient 31.
KIAA1324 has a moderate expression in ovary; nevertheless, it is known to be induced by estrogen in the female reproductive tract [
36].