Dual functions of WDR11 in the pathogenesis of hypogonadotrophic hypogonadism

Disorders of sexual development are one of the most common human birth defects affecting up to 3% of the population. Delayed or absent puberty, insufficient sex hormone production and malformation of reproductive organs - clinically termed as hypogonadotrophic hypogonadism - subsequently lead to infertility. Reproductive dysfunction is a major public health issue affecting up to 15% of couples worldwide and includes infertility, spontaneous abortion and stillbirth. Hypogonadism is the most severe form of infertility. Somewhat surprisingly, nearly 30% of infertile couples have unexplained infertility with apparently normal reproductive organs (eugonadal infertility). Pregnancy rates have improved for these patients using ovulation inducing agents along with artificial insemination or in vitro fertilisation. However, this has opened up a potential risk of transmitting abnormal genes to the next generation. In order to provide best clinical treatment and expert counselling, a better understanding of the genetics of infertility is essential.

Although some of the key genes required for the onset of puberty and development of the gonads (testis and ovary) have been identified, the underlying causes and mechanisms of the majority of infertility cases remain unexplained. This is because genes responsible for sexual development and fertility are often difficult to identify by conventional genetic studies within families, as they affect the reproductive capacity of the individual, and the sexual dysfunction is often under-recognized. Thus investigation of the genetic basis of infertility represents a major challenge in reproductive science and medicine.

To ensure normal reproductive function, proper development and coordination of the hypothalamus-pituitary-gonads (HPG axis) is essential. The key factor that regulates the HPG axis is gonadotrophin releasing hormone (GnRH) which is released by specialised neurons in the brain. Timely release of GnRH is critical for the onset of puberty and subsequent sexual maturation; thus misregulation of the function and release of GnRH will cause delayed or absent puberty, and infertility. Primordial germ cells (PGCs) are the founders of sperm and egg. Mis-regulation of PGC development during the early embryonic stage can also result in infertility due to an insufficient number of sperm or eggs present in the gonads at birth.

We have recently identified that a relatively unknown gene WDR11 is mutated in at least two forms of human reproductive disorders, Kallmann Syndrome (KS) and normosmic idiopathic hypogonadotrophic hypogonadism (nHH). These are genetic disorders defined by absent or delayed puberty and infertility, often accompanied by several other malformations in the body such as loss of sense of smell, cleft palate and kidney dysfunctions. How the mutation of WDR11 causes the disease is not known. We hypothesise that WDR11 regulates the function of GnRH neurons and PGCs. This project aims to investigate the role of WDR11 and the molecular mechanisms it may regulate during the developmental process of GnRH neurons and PGCs, defects of which may cause the clinical condition of KS/nHH.

We will employ genetically engineered mouse models that allow us to monitor the proper development of GnRH and PGCs in live embryos. Human and mouse cells in culture will also be used to study the molecular and cellular mechanisms of WDR11 and its interacting partner EMX1 that regulates gene expression. Our study will reveal new genetic basis of infertility, thus helping provide better clinical treatment, prognosis and management of the patients, as well as more accurate genetic screening and counselling.

Reproductive failure occurs in 10-15% of all couples and nearly 30% of them have unexplained infertility. Defects in the hypothalamic-pituitary-gonadal axis are likely to underlie a significant proportion of the aetiologies in these patients, but a critical barrier to proper diagnosis and treatment is the lack of understanding of the specific mechanisms of impairment. WDR11 gene was identified by positional cloning from normosmic hypogonadotrophic hypogonadism (nHH) and Kallmann syndrome (KS) patients. The mechanism of how WDR11 regulates reproductive function is not known, except that WDR11 mutations interfere with its binding to EMX1. Based on the findings of human mutations, we hypothesise that it will affect GnRH neuron migration and/or regulate GnRH secretion or action. Recently, several genes have been shown to affect not only hypothalamic function, but also directly affect gonadal function. In fact, our preliminary data suggests that WDR11 be one such protein that affects the migration of primordial germ cells (PGCs), resulting in primary gonadal defects. We hypothesise that WDR11 is important for normal hypothalamic function and PGC migration. To test this hypothesis, we will determine the phenotype of the Wdr11 KO mouse and confirm the effects of WDR11 deficiency on the development and establishment of GnRH neurons and PGCs. We will also investigate the role of WDR11 on the migratory behaviour of PGCs using Stella-GFP transgenic mouse that allows purification and live monitoring of PGCs. We will also determine the molecular mechanisms of WDR11 and EMX1 in human and mouse cell lines and test the potential involvement of Hedgehog signal pathway. These studies will reveal novel insights into the function of WDR11 in the regulation of GnRH neuronal migration and establishment of PGCs in the gonads, disruptions of which underlie human reproductive disorders.

Approximately 10-15% of all couples suffer from infertility and nearly 30% of infertile couples have unexplained infertility. Disorder of sexual development is one of the most common human birth defects affecting upto 3% of the population and remains as the main contributing factor of infertility. Kallmann Syndrome (KS) and normosmic idiopathic hypogonadotropic hypogonadism (nHH) are estimated to affect 1/4000 to 1/10,000 males and can be 2-5 time more prevalent in females. However, this figure might be an underestimate with potentially broader demographic of undiagnosed individuals with milder and isolated symptoms. Our study will directly contribute to the improved diagnosis of KS/nHH, which is often delayed and difficult due to complex and overlapping clinical symptoms. Identifying dual effects of WDR11 upon the hypothalamus and gonad are important because this information could help determine the underlying mechanisms for those who do not respond to current gonadotrophin treatment. These findings will also be relevant to eugonadal (normal) infertility patients, who could have a combination of hypothalamic and gonadal defects. Moreover, if WDR11 causes primordial germ cell (PGC) migration defects, contributing to the primary gonadal defects, it will have a significant impact on the current categorisation and management of the disease, underpinning "stratified medicine", thus potentially influences the clinical practices, health economics and public perceptions.

Definitive diagnosis and genetic counselling in the context of assisted pregnancies are central to the long-term prognosis of fertility. Although pregnancy rates have improved for patients with unexplained infertility, these treatments may be costly and result in multiple births. In addition, our study will allow better ante- and neo-natal care and pre-pubertal treatment which can improve some of the congenital defects in these patients and their future reproductive functions. It will also contribute to improve other neonatal health issues such as midline defects and sensori-neural abnormalities which are associated with KS.

Germ cells are pluripotential stem cells that can be induced to differentiate into multiple types of precursor cells highly resembling embryonic stem cells. Therefore, a better understanding of germ cell biology will enhance clinical application of stem cell technology. Defects in PGC migration underlie not only infertility but also other human disorders including childhood germ cell tumours (one of the most common neonatal tumours) that are thought to be derived from the extra-gonadal germ cells that failed to undergo proper migration into the genital ridges and were not removed by apoptosis. Therefore, the mechanisms we have already started to investigate are likely to have a considerable impact in understanding germ cell behaviour during development and in postnatal life.

Therefore, the proposed work will have substantial benefits towards the UK competitiveness and contribution to quality of life, as it will allow more accurate diagnosis and tailored treatments, improving patient management, thus reduce healthcare cost. There will be a direct link with the health care industry through stem cell applications, eventually contributing to the country's economy.

Finally, the postdoctoral fellow and the named research technician (50% post) will develop expertise in a wide-range of techniques, ranging from in vivo experimentation to cell and molecular biology. The clinical collaborations will enable them to develop significant insight for successful medical research. In particular, the named technician, Ji-Young Lee, has already showed significant potential and capacity, generating novel preliminary data using the embryo slice culture techniques, thus this grant will provide a critical opportunity for her development in academic research career.