Mutation analysis of the WNT4 gene in Han Chinese women with premature ovarian failure

Premature ovarian failure (POF) is a rare disease that severely affects the reproductive health and endocrine balance of 1% of all women [1]. It is characterized by abnormal follicular genesis, development or apoptosis. POF presents clinically as primary amenorrhea in women with abnormal prepubertal onset, or as spontaneous secondary amenorrhea in women with premature depletion of ovarian follicles/arrested folliculogenisis. The etiological factors and pathogenesis of POF are extremely heterogeneous. As a severe complication in patients after treatments for ovarian neoplasms or autoimmune diseases, the damage caused by iatrogenic factors (surgery, chemotherapy, radiations) accounts for many cases. However, the causes are still unclear in most cases of POF (especially idiopathic form). Genetic factors are known to provide a significant etiological component in recent studies. Apart from studying X chromosome deletions and X/autosome translocations in patients, research has focused increasingly on some candidate genes involved in ovarian failure in animal models, such as Fshr and Foxl2 [2].

Wnt4, which encodes a secreted extracellular signaling protein, is known to be involved in multiple developmental processes, such as the formation of the kidney, adrenal gland and gonads [3‐5]. Many studies have demonstrated that this gene plays an important role in mammalian female sexual differentiation. It is downregulated in the testis and upregulated in the ovary at 11.5 days post-coitum in the mouse by activating Frizzled-4, expressed on luteal cell membranes to activate distinct signaling cascades [5]. The inactivation of Wnt4 led to ovarian dysgenesis with early production of testosterone by mesenchymal cells and formation of male internal genitalia, including testicular tubules and spermatogonia in XX mice [6, 7].

Recently, attention has been paid to the contribution of WNT4 for the maintenance of ovaries and survival of follicles in the mouse and humans. WNT4 is expressed in the bipotential embryonic gonad of both sexes, and increases when small primary follicles are formed during mid-pregnancy. Vainio et al. observed that the number of oocytes in Wnt4 mutant mouse ovaries was abnormally low at birth [5]. Although Wnt4 has not been shown to affect the number of primordial germ cells in the undifferentiated gonad, it is thought to act as an oocyte survival factor during female embryogenesis [8]. Compared with wild-type mice, Wnt4 deficient mice had a markedly enhanced rate of oocyte apoptosis [9, 10]. There are two oocyte-specific transcriptional factors, FIGLA and NOBOX, acting downstream of WNT4 signaling, which are associated with POF [11, 12]. Therefore, we carried out mutational screening of the WNT4 gene in Han Chinese women with POF.

Apart from the accepted view that WNT4 is a key regulator of gonadal determination and differentiation, its mRNA and protein are present in human ovaries during fetal development and at different stages of follicular development in adult ovaries [10]. Some functional analyses revealed that deficiencies in the WNT4 gene might affect the development of ovaries and follicles. Vainio et al. observed that the ovaries of Wnt4 mutant female mice had only a few degenerating oocytes [5]. Yao et al. found that germ cells in the ovarian cortex were almost completely absent in both Wnt4 and Fst null gonads and concluded that Wnt4 might play a critical role in maintaining germ cell survival in the ovary [9]. Similarly, Jääskeläinen et al. also revealed that Wnt4-deficient mice had a markedly enhanced rate of oocyte apoptosis [10].

Based on the above-mentioned studies and views, researchers began to study this gene from different aspects in human diseases, mainly involving Müllerian Duct abnormalities. A French collaborative study identified a new L12P mutation with Exon 1 of the WNT4 gene from a patient who presented with uterine hypoplasia and follicle depletion [13]. Biason-Lauber et al. also found that one girl with absence of a uterus, androgen excess and lack of follicles in her left ovary carried a novel loss-of-function dominant negative mutation in WNT4 [14]. Moreover, a homozygous missense mutation in the human WNT4 gene was identified in a novel autosomal-recessive syndrome SERKAL which consists of female to male sex reversal and renal, adrenal, and lung dysgenesis and is associated with additional developmental defects [15]. Therefore, we tried to determine whether Han Chinese women with POF might show a genetic background linked to the WNT4 gene. However, we did not observe any causal variant: only a silent mutation. Wnt genes have been highly conserved throughout evolution and play pivotal roles in a wide variety of physiological events during embryogenesis. We supposed that this conservatism and importance might determine the stability of the WNT4 gene. In addition, Jääskeläinen also pointed towards species-specific differences in the expression patterns of Wnt4 when comparing the results from mice and humans [10].