Mutations in inhibin and activin genes associated with human disease

Molecular and Cellular Endocrinology 359 (2012) 113–120

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Review

Mutations in inhibin and activin genes associated with human disease Andrew N. Shelling ⇑ Department of Obstetrics and Gynaecology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand

a r t i c l e

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Article history: Available online 30 July 2011 Keywords: Inhibin Activin Premature ovarian failure Ovarian cancer Genetic disease

a b s t r a c t Inhibins and activins are members of the transforming growth factor (TGFb) superfamily, that includes the TGFbs, inhibins and activins, bone morphogenetic proteins (BMPs) and growth and differentiation factors (GDFs). The family members are expressed throughout the human body, and are involved in the regulation of a range of important functions. The precise regulation of the TGFb pathways is critical, and mutations of individual molecules or even minor alterations of signalling will have a significant affect on function, that may lead to development of disease or predisposition to the development of disease. The inhibins and activins regulate aspects of the male and female reproductive system, therefore, it is not surprising that most of the diseases associated with abnormalities of the inhibin and activin genes are focused on reproductive disorders and reproductive cancers. In this review, I highlight the role of genetic variants in the development of conditions such as premature ovarian failure, pre-eclampsia, and various reproductive cancers. Given the recent advances in human genetic research, such as genome wide association studies and next generation sequencing, it is likely that inhibins and activins will be shown to play more important roles in a range of human genetic diseases in the future. Ó 2011 Elsevier Ireland Ltd. All rights reserved.

Contents 1. 2.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of mutations and disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Mendelian disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Complex disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Other genetic mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inhibin and activin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Functions of inhibin and activin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Knockout mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diseases associated with mutations in the inhibin and activin genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Reproductive disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1. Premature ovarian failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.2. Pre-eclampsia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1. Ovarian cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2. Adrenal tumours. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.3. Prostate cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.4. Testicular germ cell tumours . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.5. Summary of inhibins and activins and cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. INHA and twinning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transforming growth factor b superfamily signalling pathway and human disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future developments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

⇑ Tel.: +64 9 3737 599x83504; fax: +64 9 3737 677. E-mail address: [email protected] 0303-7207/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2011.07.031

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1. Introduction Inhibins and activins are members of the transforming growth factor (TGFb) superfamily, that contains more than 30 structurally related polypeptide growth factors. The family also includes the TGFbs, bone morphogenetic proteins (BMPs) and growth and differentiation factors (GDFs) (Stenvers and Findlay, 2009), and will be discussed in more detail in other parts of this special issue. The majority of family members are ubiquitously expressed, and nearly every cell in the body will express and respond to at least one member of the superfamily (Gordon and Blobe, 2008). Members of the superfamily are involved in the regulation of a range of important functions, including proliferation, differentiation, development, and angiogenesis. The precise regulation of the TGFb pathways in each of these important biological functions is critical. This is supported by the finding that mutations in individual molecules, or even minor alterations of signalling, will significantly affect ligand function. In many situations this may lead to the development of disease or predisposition to the development of disease (Ikushima and Miyazono, 2010). This review will begin with an overview of what we currently understand in the role of mutations and gene variants in human disease, and then discuss specifically how mutations or variants in the inhibins and activins have been implicated in human disease. 2. Role of mutations and disease In traditional genetic studies, most of the focus is on single gene or Mendelian disease, that tend to be rare, severe and observed with clear patterns of inheritance. However, there is increasing interest in the more common diseases, often referred to as multifactorial or complex diseases (Frazer et al., 2009). In reality, there is a continuum between single gene and complex disorders, with different numbers of genes being involved, along with variable contributions of environmental factors. Different approaches are required to identify genes involved in each of these different types of genetic disease. 2.1. Mendelian disease For Mendelian disease, specific genes are the focus of attention, and gene mutations are identified within individual patients and families. Often this involves linkage analysis using large pedigrees, which is sometimes successful in finding the causal gene. Even once the putative causal gene is identified, it is often difficult to show it really is the disease-associated gene. Sometimes the gene is not fully penetrant in families, that is, not all gene carriers are actually affected. Often, to be convinced that a given gene and mutation in that gene are involved in the disease, large numbers of patients with a clearly defined phenotype need to be studied, and compared to normal controls. In addition, functional models, such as knockout mice or the expression of mutant proteins in human cell lines, are required to confirm the role of the mutation. 2.2. Complex disease There is now increasing interest in the role played by genetic predisposition and susceptibility in common complex diseases, such as cancer, heart disease, diabetes, and infertility (Shelling, 2009). Many of these disorders show familial aggregation suggestive of a genetic predisposition, however, they may also present with weak family histories, without any clear patterns of inheritance. This can be problematic if there are also strong environmental factors also involved in the aetiology of the disease. It can be difficult to separate the role of genetic causes from the affects of

common environmental factors such as smoking, obesity, diet and exposure to exogenous hormones. It is now clear that genetic predisposition is due to minor variations in a large number of genes, neither one alone being sufficient to cause the disorder. These variants are not mutations, as by themselves they are not capable of causing the disease, but are termed polymorphisms, as they are genetic variants that are markers of disease susceptibility. Some of these polymorphisms are single nucleotide polymorphisms, or SNPs, which can be measured at the genome level using technologies such as a SNP array. The data generated from SNP arrays has allowed for genome wide association studies (GWAS), and led to the association of gene variants with the likelihood of developing disease. 2.3. Other genetic mechanisms As we understand more about the human genome, the observation has been made that gene mutations are not the only cause of genetic disease. Other genetic mechanisms must also be involved, and we are currently beginning to discover and understand some of these. The human genome is more complex than we ever expected, and genes can be regulated in many ways, not just by gene mutations. Epigenetic mechanisms are those that are not due to changes in the DNA sequence of genes, but have been shown to be involved in the development of disease, primarily through their ability to alter the level of gene expression (Moss and Wallrath, 2007). Genomic imprinting is one example of epigenetics, whereby genes are marked according to their parental origin, and results in monoallelic expression. The imprint mark is often methylation of CpG-rich domains that are established during gametogenesis. Copy number variants are also important and significant causes of disease (Shelling and Ferguson, 2007). Copy number variants include events such as gene duplication, deletions and other large scale rearrangements, and may play a role in regulating the change in the level of expression of nearby genes. These types of genetic variation have only recently been discovered, or more correctly, their significance has only recently been realised, and appear to be an important driver of human genomic evolution. Even more recent is the discovery that microRNAs, which are small non-coding RNAs, are important in the pathogenesis of many human diseases (Nicoloso et al., 2010). By binding to the 30 untranslated region (30 UTR) of an mRNA, the miRNA targets it to be translationally repressed or degraded, with one miRNA potentially interacting with hundreds of target gene products. 3. Inhibin and activin Activin and inhibins are dimeric glycoprotein hormones with opposing actions in the hypothalamic pituitary axis and gonads. Much of their biology will be described in other manuscripts in this special issue. Inhibins are made up of a common a subunit linked to a b subunit (most commonly bA and bB), to give inhibins A and B. Activins are homodimers of two b subunits (most commonly bA and bB) forming activin A, activin B or activin AB isoforms. Additional b subunits isoforms (bC and bE) have been identified in the human (Lau et al., 2000), and it has been proposed that activin C is an antagonist of activin A bioactivity (Gold et al., 2009). The a, bA and bB subunits are encoded by three distinct genes (Barton et al., 1989). The genes encoding the a and bB subunits (INHA and INHBB) are located on chromosome 2. The inhibin a locus is 2q33-36 while INHBB is located at 2cen-q13. The INHBA locus is on chromosome 7p15-p13 (Barton et al., 1989). Members of the TGFb family form complexes with specific type I and II receptors. Signalling is initiated upon the binding of activin to two cell surface serine/threonine kinase receptors, the type II

A.N. Shelling / Molecular and Cellular Endocrinology 359 (2012) 113–120

receptor (either ACTRIIA or ACTRIIB) and, subsequently, the type I receptor (ActRIB). The receptors are phosphorylated, which leads to the activation of receptor specific SMAD molecules. The signal is then transduced within the cell via the SMAD2, SMAD3 and SMAD4 transcription factor proteins. 3.1. Functions of inhibin and activin The importance of inhibins and activins in the regulation of the female reproductive cycle is well established (recently reviewed by Stenvers and Findlay, 2009). Inhibins have largely an endocrine role, being produced mainly by the gonads, and providing negative regulation of pituitary follicle stimulating hormone (FSH) synthesis and secretion from the anterior pituitary. This is most obviously seen following castration in either males or females that results in the loss of circulating inhibin, and a rise in serum FSH. Inhibins also have a local (paracrine and autocrine) action. Activins are expressed in a wide range of tissues outside of the ovaries and testes, including the placenta, pituitary, adrenals, spleen, bone marrow and specific regions of the brain (Luisi et al., 2001). This suggests that activins may have a greater range of functions than the inhibins, where they are acting as paracine and autocrine growth factors. The ovaries and the testis are the major sites of inhibin and activin production. In reproductive women, the ovarian granulosa, theca and luteal cells express the inhibin a subunit, bA and bB subunits. The major sites of inhibin a subunit production are the granulosa and theca interna cells and are consistently overexpressed within the follicle relative to b subunits. In females, inhibins and activins are known to act locally in the ovary, and play opposing roles. The balance of expression between inhibins and activins produced within the granulosa cells are important in regulating a range of functions associated with follicular development, including granulosa cell maturation and proliferation, thecal cell androgen synthesis, and oocyte support and development (Knight and Glister, 2006). In males, inhibins and activins play an endocrine role in regulating FSH levels, but are also thought to also have paracrine and autocrine role in regulating Leydig and Sertoli cell proliferation, differentiation and steroidogenesis (Stenvers and Findlay, 2009). Most of the effects of inhibin are associated with its antagonism of activin signalling. This can be seen both at the endocrine level of regulating FSH release from the anterior pituitary. It is also likely that inhibin antagonises activin action at the paracrine and autocrine level, as they have been shown to have opposing roles in steroidogenesis, gamete development and specific cell proliferation within reproductive tissues. Inhibin can also antagonise activin action through its ability to bind to betaglycan, a non-signalling coreceptor for TGFb, which prevents activin signalling by preventing the formation of a functional activin receptor complex (Lewis et al., 2000; Bilandzic and Stenvers, 2011). In a similar way, follistatin is an activin binding protein that blocks the ability of activins to bind to its receptors (Findlay et al., 2002). 3.2. Knockout mice A common way to determine the role and function of a particular gene is to create a knockout mouse, and determine what the phenotype of the mice are. Although animal models do not always exactly reflect human disease, as many gene pathways have evolved differently across species, knock out mice still serve as one of the best in vivo models available to assess the contribution of specific genes in a given disorder. The importance of inhibin in reproductive biology is clearly seen in mice with a homozygous deletion of the inhibin a subunit gene (Matzuk et al., 1992). The Inha/ mice develop gonadal sex

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cord tumours within 6 weeks, causing death in males and females in 12 and 17 weeks, respectively, which suggests that inhibin may act as a tumour suppressor gene. These tumours develop with 100% penetrance, and are seen as Sertoli cell tumours (in males) and granulosa cell tumours (in females). The development of gonadal cancer is followed by a cancer cachexia-like wasting syndrome that is associated with weight loss and stomach and liver related problems (Matzuk et al., 1994). When the knockout mice were gonadectomised, life expectancy increased, however, these mice developed adrenal tumours that caused death at 36 and 33 weeks in males and females, respectively. Activin levels were elevated in these mice, and several lines of evidence indicate that the high levels of circulating activin may be have led to cachexia from 6 to 7 weeks and lethality by 12 weeks (Matzuk et al., 1994). The tissue-specific responses observed in cell growth characteristic of the adrenal and gonads highlight tissue-specific functions of inhibins and activins. Recent studies have shown that the antagonism of ActRIIB can lead to a reversal of cancer cachexia and muscle wasting, suggesting a promising new approach for treating cachexia (Zhou et al., 2010). 4. Diseases associated with mutations in the inhibin and activin genes 4.1. Reproductive disorders Given that a variety of members of the TGFb superfamily regulate the male and female reproductive system, it is not surprising that most of the diseases associated with abnormalities of the inhibin and activin genes are focused on reproductive disorders and reproductive cancers. 4.1.1. Premature ovarian failure Premature ovarian failure (POF) is characterised by ovarian dysfunction leading to a menopause-like state in women earlier than 40 years of age (recently reviewed by Shelling, 2010). It is a common cause of infertility, and affects one in 100 women under 40 years and one in 1000 women under 30 years of age (Coulam et al., 1986). A positive family history is reported in up to 30% of women with POF. Serum levels of FSH above 40 IU/l and amenorrhoea for duration of 6 months or more are the clinical parameters defining POF. Additionally, affected women have very low levels of circulating oestrogen. Clinical symptoms observed are similar to those observed with the onset of natural menopause, such as hot flushes, vaginal dryness, dyspareunia, insomnia, vaginitis and mood swings. In addition, psychological problems may result from the realisation that fertility is no longer possible. There are elevated risks for low bone density (osteoporosis) due to the low levels of oestrogen experienced by these women. The aetiology of POF is heterogeneous with the majority being idiopathic. Known causes of POF include permanent damage to the ovaries, such as pelvic surgery, chemotherapy or radiotherapy, autoimmune conditions, exposure to environmental toxicants and genetic causes (recently reviewed by Persani et al., 2010; Shelling, 2010). In women experiencing a normal menopause, a decline in serum inhibin levels occurs at about age 40, when the stores of ovarian follicles begins to diminish below a certain threshold of numbers of follicles (MacNaughton et al., 1992; Richardson et al., 1987). This decline in circulating inhibin levels results in raised FSH concentrations, and increased follicle recruitment for the remaining few years of reproductive life. In addition, women with POF have low serum levels of inhibin A and inhibin B, compared to age-matched fertile women (Munz et al., 2004). Therefore it is a plausible hypothesis that inhibin is a good candidate marker for the development of POF. It is proposed that a functional mutation

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in any one of the three inhibin genes could lead to a decrease in the amount of bioactive inhibin (Shelling et al., 2000). This loss could remove the negative feedback on the pituitary, cause an increase in FSH levels contributing to premature depletion of follicles and hence result in POF. Mutational screening of the inhibin a subunit gene (INHA) identified a missense mutation INHA G769A causing an amino acid transition from alanine to a threonine at amino acid position 257, Ala257Thr (Shelling et al., 2000). In this initial study on a New Zealand population, a heterozygous change was identified, significantly correlated to the condition (7% in POF, n = 38 compared to 0.07% in controls, n = 150; p = 0.011). Significant mutations were not found in the inhibin bA or bB subunit genes, although a silent transition 1032C>T variant was observed in the INHbA gene (Shelling et al., 2000). Several subsequent studies have been undertaken (Marozzi et al., 2002; Jeong et al., 2004; Dixit et al., 2004, 2006; L’vshits et al., 2005; Sundblad et al., 2006; Corre et al., 2009; Prakash et al., 2010). Most, but not all studies have confirmed this relationship (see Table 1, reviewed by Chand et al., 2010; Zintzaras, 2010). A single large recent study indicates that the association is not significant in Italian and German women with POF (Corre et al., 2009). Ethnic differences are also likely to be important. Given that there was some debate in the literature about the role of the INHA G769A in POF from genetic data, it was important to provide further proof that the variant was associated with POF, therefore a functional study was performed (Chand et al., 2007). Wildtype and mutant forms of the inhibin a subunit gene were constructed. An activin sensitive luciferase reporter construct was transfected into cell lines, including a biologically relevant gonadotroph cell line (LbT2), a granulosa cell tumour cell line, (COV434), and an embryonic kidney cell line (HEK293), in the presence of either wild-type or mutant inhibin a subunit. The overexpression of wild-type inhibin a subunit resulted in a dosedependent decrease in expression of the luciferase reporter activity in all three cell lines. In contrast, activin-induced reporter activity was unaffected by increasing doses of inhibin a mutant DNA. This

was shown to be due to a direct affect on activin action, and was not to reduced dimer formation (Chand et al., 2007). This region of the protein is also highly conserved, suggesting that it may be an important regulatory region, and variation through this region during evolution has not been tolerated. Overall, this functional data would suggest that in this model system, the mutant inhibin is less bioactive (Chand et al., 2007), and therefore supporting the earlier genetic association studies. Further functional proof has come from a gene knockout study (Myers et al., 2009), that showed that the loss of the inhibin a subunit in the knock out mouse is important for early folliculogenesis. The loss of inhibin a led to the gain of unopposed activin, increasing granulosa cell derived activin signalling, which led to precocious follicular development. One of the important conclusions from this research was that the loss of inhibin a does not change the number of primordial follicles initially present in the neonatal mouse, but later led to an acceleration of follicle growth from the resting pool into the growth phase. This represents a good model for what was originally hypothesised would occur in women with an inhibin a gene variant with reduced activity (Shelling et al., 2000). During mutational analysis of the inhibin a gene, Marozzi et al. (2002) noted an association between a promoter SNP (16C>T) with POF, which has since been confirmed (Harris et al., 2005). Other SNPs were identified (124A>G and 252C>A) and a repetitive region consisting of an extended imperfect TG repeat at approximately 300 bp which is considerably polymorphic in both the POF patient and control populations, with the repeat region ranging from 76 to 94 bp in length (see Fig. 1). Certain haplotypes of the SNPs and repeat were seen to be associated with POF (Harris et al., 2005; Woad et al., 2009), but results were not consistent and seemed to show differences between ethnic groups. Other studies have not found a significant association between the 16C>T SNP and POF (Sundblad et al., 2006; Dixit et al., 2006; Corre et al., 2009). It is unclear whether these polymorphisms in the INHA promoter might result in reduced inhibin expression, but suggests that promoter polymorphisms may be another mechanism for the

Table 1 Reported frequencies of INHA 769G>A gene variants. The patients from the Marozzi et al. (2002) study are included in the Corre et al. (2009) study. Population studied

POF patients with variant

Control samples with variant

Publication

New Zealand India Korea Argentina India Italy and German Total

7/166 9/80 0/80 1/59 13/100 21/611 51/1096 (4.7%)

1/150 0/100 0/100 8/149 2/50 76/1084 87/1633 (5.3%)

Shelling et al. (2000), Woad et al. (2009) Dixit et al. (2004) Jeong et al. (2004) Sundblad et al. (2006) Prakash et al. (2010) Corre et al. (2009)

TG repeat ~-300bp

-252C>A

Promoter

-124A>G

G769A

-16C>T

Exon 1

Exon 2

Pro

-N

-C

Fig. 1. Representation of the Inhibin alpha gene, and position of the variants identified in POF patients, as reported by Shelling et al. (2000), Marozzi et al. (2002), Jeong et al. (2004), Dixit et al. (2004, 2006), Harris et al. (2005), L’vshits et al. (2005), Sundblad et al. (2006), Woad et al. (2009), Corre et al. (2009), Prakash et al. (2010). The human INHA gene has two exons spanning a 1.7 kb intron. The precursor has three regions, pro-, aN and aC. The aC is the mature inhibin alpha subunit. The gene and promoter region are not drawn to scale.

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transcriptional regulation of the inhibin a subunit. Polymorphisms in genes that regulate the rate of decline of the follicular pool during reproductive life have been proposed to be the same genes that may be responsible for the variation in the age at menopause (Te Velde and Pearson, 2002). The size of the upstream repeat region has expanded during evolution in mammals, and it is interesting to note that the shortest repeat appears to be protective against early ovarian aging (Harris et al., 2005). Further studies are required in larger population samples to confirm the association between mutant INHA G769A, INHA SNPs and POF. While the reduction of inhibin bioactivity by the INHA G769A mutation is clearly not the only cause of the condition, evidence suggests that this change may serve as a susceptibility factor, increasing the likelihood of POF. Other predisposing factors including other genes, ethnicity and lifestyle factors will likely play a role, but at the moment, the roles of these factors remain largely unknown. While the functional studies support the association, we need better understanding of how the mutant inhibin acts to bring about early loss of follicles, either through its inability to reduce FSH levels, or by paracrine actions that occur within the granulosa cell. 4.1.2. Pre-eclampsia Pre-eclampsia (PE) is a condition of pregnancy-induced hypertension associated with significant levels of protein in the urine. It occurs in as many as 10% of all pregnancies. One feature of PE is elevated maternal serum inhibin A in the second and third trimester of pregnancy. This might suggest that inhibin gene variants leading to elevated levels of inhibin A, may be involved in the disease process. Therefore, Ciarmela et al. (2005) looked for mutations in INHA in a group of 50 women with PE. They found the Ala257Thr variant was not at increased frequency in their PE patients (1/50; 2%) compared to controls (10/145; 7.6%). The frequency of the variant in controls is markedly different to other studies. It is clear that the INHA Ala257Thr variant is not a cause of PE. The authors suggest that the INHA Ala257Thr variant may be more likely to be a polymorphism in the Italian population. 4.2. Cancer Several lines of evidence point toward the role of inhibin a as a tumour suppressor gene (Stenvers and Findlay, 2009). This is primarily due to the cancer phenotype of the INHA knockout mouse, as has previously been discussed (Matzuk et al., 1992). In addition, human granulosa cell tumours (GCT) and other types of ovarian cancer are associated with elevated levels of circulating inhibins (Healy et al., 1993). This seems counter-intuitive, as tumour suppressor genes normally function through loss of gene expression by mechanisms such mutation, deletion, or methylation. That inhibin might be overexpressed in some tumours creates a dilemma in our understanding of the aetiology of cancer in general, as overexpression is normally the hallmark of an oncogene. That raises the possibility that cancer arises due to a defect in the activin signalling pathway, leading to elevated levels of inhibin, due to the development of resistance (Fuller and Chu, 2004). The activin signalling pathway appears to be growth-suppressive (Ramachandran et al., 2009) in the same way that the TGFb signalling pathway plays a significant role in growth suppression in a range of normal epithelial cells. One explanation is that a reduction in TGFb signalling in tumour cells is often accompanied by increased secretion of the ligand (de Caestecker et al., 2000) and/or downregulation of receptors (Kalkhoven et al., 1995), and leads to elevated circulating TGFb levels in cancer (Bernard et al., 2001). This could benefit the cancer through production of extracellular matrix, suppression of immune system function, or promotion of angiogenesis. Activin resistance, like that proposed in the

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TGFb pathway, may lead to the loss of growth inhibitory affects, and also the increased secretion of inhibin (and presumably activin) at high levels in the serum. One proposed mechanism for the loss of responsiveness of ovarian tumour cells to the action of activins is through down-regulation of betaglycan expression. Many tumours show loss of betaglycan expression (reviewed by Gatza et al., 2010), including granulosa cell tumours (Fuller and Chu, 2004), however relationships between inhibins, activins and other members of the TGFb superfamily are complex. However, the development of resistance to activin mediated growth suppression, and associated elevated inhibin, may be an important stage of cancer development. In the development of cancer, complex chromosomal abnormalities are frequently seen. It is difficult to associate some of the specific karyotypic changes with particular types of cancer, and it appears that many of these chromosomal changes are purely random events. Chromosome regions with tumour suppressor genes may show deletions or loss of heterozygosity, whereas those regions with oncogenes may show amplification or other rearrangements. There is speculation as to whether these chromosomal abnormalities play a significant role in tumourigenesis, or whether they are random events related to increased cell division. Risbridger et al. (2001) reviewed the chromosomal regions of the inhibin and activin subunit genes, and noted that some of these regions were altered in endocrine tumours, but many of these appear to be close to background levels of alteration, and therefore do not specifically implicate them as tumour suppressor genes. 4.2.1. Ovarian cancer There is a well established link between human ovarian cancer and inhibin, however, to date, no mutations have been found in either the inhibin or activin genes. Several studies have shown that women with Granulosa Cell Tumours (GCT’s) and mucinous ovarian tumours have elevated serum levels of inhibin (Healy et al., 1993). Immunoreactive assays to the a-subunit detected increased levels of inhibin in postmenopausal women in 100% GCT and 90% mucinous tumours (Robertson, 1999). Elevated levels of total inhibin in sera are useful as a diagnostic marker for GCT in these women (Robertson, 2002), and are a feature that has been used to detect relapse in some patients before clinical manifestation of tumour recurrence. If inhibin is acting as a tumour suppressor gene in ovarian cancer, then we might expect that the chromosomal region harbouring inhibin a may frequently undergo loss of heterozygosity (LOH). However, given that only one of 17 GCT showed LOH in the vicinity of inhibin a gene, this would suggest that it is not acting as a classical tumour suppressor gene in that tumour subtype (Watson et al., 1997). LOH was observed in 12/36 (33.3%) of epithelial ovarian tumours (Watson et al., 1997) and 1/22 in another study (Depasquale et al., 2002), which suggests that loss of heterozygosity of inhibin a is not a common mechanism for tumour development in epithelial ovarian cancer cases. Similarly, Depasquale et al., 2002 found no loss of heterozygosity at the inhibin/activin bA subunit on chromosome 7p. 4.2.2. Adrenal tumours Given that inhibin a has been known to be produced in the adrenal glands, and that the inhibin a knock out mouse develops adrenal tumours when gonadectomised, it would be reasonable to suspect that adrenal tumours may also show abnormalities of INHA. While no chromosomal abnormalities are reported, immunohistochemical studies reported increased immunoreactivity (Pelkey et al., 1998). However, a study in 39 patients with paediatric adrenocortical tumours found several polymorphisms, and three mutations/variants in INHA, from blood DNA (Longui et al., 2004). Those germline mutations were Pro43Ala (one patient),

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Gly227Arg (three patients) and Ala257Thr (two patients). Two of these variants were newly identified (Pro43Ala, Gly227Arg), while the other (Ala257Thr) has been previously identified in POF patients (Shelling et al., 2000). The authors (Longui et al., 2004) suggest that these variants may predispose to adrenocortical tumours, however, the study was small, and does not appear to have been replicated in other populations. It is interesting to note that two of the variants (Pro43Ala, Gly227Arg) have not be found in other studies of cancer and reproductive disease, and their significance is currently unknown. 4.2.3. Prostate cancer Prostate cancer is a common malignancy of males. The normal growth and function of the prostate gland is dependent on androgens, but also members of the TGFb superfamily. It has been shown that there is loss of inhibin a subunit expression in high-grade prostate cancer (Mellor et al., 1998), and evidence that promoter methylation and loss of heterozygosity is associated with the down-regulation of the INHA in prostate cancer cell lines, which is consistent with its tumour suppressive role (Balanathan et al., 2004). 4.2.4. Testicular germ cell tumours Given that the INHA knockout mouse developed both testicular and ovarian stromal tumours with near 100% penetrance, Purdue et al. (2008) looked for SNPs that might be associated with Testicular Germ Cell Tumours. They identified several SNPs in the inhibin and activin genes, INHA, INHBA, INHBB, INHBC, and INHBE, as well as SMAD4, and looked for association in 577 Testicular Germ Cell Tumours samples and 707 matched controls. They found one SNP in INHA that appeared to be significantly associated, but could not find association with other SNPs. The associated SNP was approximately 2 kb downstream from the INHA stop codon. Purdue and colleagues considered that given its location, it was unlikely to be a causal SNP, but suggested that it could be linked with a causal SNP, although they were unable to find one. They also considered that it was unlikely to be associated with the INHA Ala257Thr (rs12720062) variant in exon 2, and was more likely to be associated with mRNA stability or translational efficiency. It is now considered that some gene variants that are located within the 30 untranslated region of a gene are sometimes associated with abnormal miRNA binding (Nicoloso et al., 2010). 4.2.5. Summary of inhibins and activins and cancer Abnormalities in the levels of inhibin and activin are clearly seen in ovarian cancer and possibly some other cancers, and these appear to correlate with tumour development. No consistent mutations have been identified in cancer patients, but other mechanisms such as methylation may be a reason for causing altered expression. There are several mechanisms by which INHA might act as a tumour suppressor gene, and it is possible that some of these might be operating at the same time. First, it might be considered that the inhibin a subunit may act as a tumour suppressor protein by dimerising with the b subunits. Thus, the normal role of inhibins may be in preventing activin formation, with a subsequent decrease in activin signalling activity. Therefore, the loss of inhibin function in cancer, may lead to an increased action of activin by unopposed subunit assembly. Second, inhibin acts directly as a typical tumour suppressor gene by inhibiting activin signalling in epithelial cells. In most cases, activin acts in the opposite manner to inhibin, for example, inhibin increases androgen production of theca cells in vitro, while activin has an opposite effect. In addition, activin, known to be involved as a growth factor in a wide range of cells, is thought to promote the proliferation of human granulosa cells in vitro. Third, as FSH is mitogenic for granulosa cells, loss of

inhibin (and/or gain of activin) may provide an elevated systemic FSH level, leading to increased proliferation of FSH-responsive cells and eventual transformation (Burger and Fuller, 1996). Both granulosa cell hyperplasia and elevated FSH levels precede tumour development in inhibin a knockout mice (Matzuk et al., 1992). 4.3. INHA and twinning Natural multiple pregnancies, or twinning, is familial, suggesting a genetic predisposition. Women who have dizygotic twins have raised levels of FSH, and inhibin is known to regulate levels of FSH. Therefore, given that inhibin regulates FSH levels, it was plausible that inhibin a might be a good candidate gene for causing dizygotic twinning. In addition, animal studies have shown that immunisation against the inhibin a subunit results in increased ovulation rates (Medan et al., 2004). Montgomery et al. (2000) identified two SNPs (16C>T, 530C>T) at the inhibin a locus, but could not show linkage between the INHA locus and dizygotic twinning in 1125 individuals. One of these SNPs, 16C>T, was implicated in POF (Marozzi et al., 2002; Harris et al., 2005). The link between POF and twinning is interesting, as both appear to involve a mechanism with increased ovulation rates, therefore it is not surprising that they may both related and caused by the same gene(s). Indeed, other members of the TFGb superfamily have been known to be important in twinning, and have also been studied in women with POF, with the identification of the BMP15 Y235C variant in an Italian patient with POF (Di Pasquale et al., 2004), but mutations were not identified in either BMP15 or GDF9 in New Zealand (Chand et al., 2006) or the Japanese population (Takebayashi et al., 2000), which suggests that they are a rare event. 5. Transforming growth factor b superfamily signalling pathway and human disease Other members of the TGFb superfamily have been associated with a range of disorders and several good recent reviews have outlined their role in disease (Gordon and Blobe, 2008; Harradine and Akhurst, 2006). Germline mutations in several members can lead to a diversity of inherited disease, including reproductive, cardiac, skeletal, muscular and connective tissue disorders (Gordon and Blobe, 2008; Harradine and Akhurst, 2006). Most of these inherited disorders are typically autosomal dominant conditions, but also show variable penetrance and expressivity. This might be expected from a tightly regulated pathway, that often shows some redundancy in gene function. Connective tissue disorders, such as Marfan and Marfan-like disorders have been associated with loss of function TGFBR1 and TGFBR2 mutations (Akutsu et al., 2007). Various other skeletal, muscular and developmental disorders have also been associated with other members of the TGFb superfamily, including the BMP and GDF families. Reproductive disorders, apart from POF as previously described, include the Persistent Mullerian Duct syndrome. This condition is usually seen when a male presents who has a uterus and sometimes other Mullerian duct derivatives, along with undescended testis who is otherwise genotypically and phenotypically normal. It is a congenital disorder, usually due to mutations in either the Anti-Mullerian Hormone (AMH) gene, or its receptor (AMHR), both giving similar phenotypes (Imbeaud et al., 1995). Both homozygous and heterozygous mutations have been identified, which suggests that haploinsufficiency may play a role in the disorder, whereby the loss of one allele is also sufficient to produce the phenotype, and is therefore an example of incomplete or partial dominance. Germline mutations in members of the TGFb superfamily are also seen in some inherited cancer syndromes. Juvenile polyposis

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syndrome is due to germline mutations in either SMAD4 (approximately 20% of cases) or BMPR1A (20%) (Howe et al., 2004). Hereditary non-polyposis colorectal cancer (HNPCC) is due to germline mutations in mismatch repair genes that cause instability of repetitive gene sequences such as microsatellites, throughout the entire genome. Genes that have a repetitive sequence are at risk of being affected by changes in the number of repeats. The TGFBR2 gene has a 10 base pair polyadenine repeat sequence that is altered in approximately 80% of HNPCC patients (Gordon and Blobe, 2008). Sporadic tumours will also need to find a way to overcome the repressive nature of this pathway. In most cell types, TGFb is a potent inhibitor of cell growth, arresting cell cycle progression in late G1 phase. Critical mutations in the TGFb pathway also lead to the loss of the ability of the cell to undergo apoptosis. Diminished responsiveness to TGFb signalling is a common feature of sporadic cancers such as breast, colorectal, ovarian, lung, pancreatic and prostate, although other cancers will undoubtedly also be involved (Francis-Thickpenny et al., 2001). Betaglycan is an important molecule in mediating the action of inhibins and activins, which are essential for a range of reproductive functions. Inhibins can bind to activin type II receptors (ActRII), and prevent the recruitment of activin type I receptors, thereby antagonising the action of activins (Lewis et al., 2000). Inhibins are most effective at this antagonist activity, when betaglycan is present. By themselves, betaglycan does not have a signalling role, but acts as a non-signalling cofactor for activins, and other members of the TGFb superfamily. It has also been shown to be localised in a range of tissues mainly those of reproductive and endocrine function, consequently it has been shown to be an important regulator of reproduction and cancer (reviewed by Bilandzic and Stenvers, 2011). Therefore it is likely to be a good candidate gene as a site of mutation in cancer and reproductive diseases. To date, no mutations have been identified, perhaps suggesting that is must play an essential role in embryological development, but two small studies in POF patients (Dixit et al., 2006; Chand et al., 2007) have identified the presence of a number of SNPs in betaglycan, while some of these SNPs were significantly associated with the development of POF, given that they were small studies, their causative role remains unclear, and remain as rare variants of unknown significance.

6. Future developments A greater understanding of the roles of inhibins and activins in various human diseases has occurred over the last few years, and many new associations have only recently been made. Much attention in the past has been on the endocrine conditions and clinical roles of circulating levels of inhibins and activins in normal reproduction and reproductive disease. The future may see a greater analysis of autocrine and paracrine affects of abnormal levels or mutant forms of inhibins and activins, and also an investigation of roles in non-reproductive conditions. For genetic research to understand more about the role of inhibins and activins in human disease, there will be increasing focus on the analysis of the entire genome in disease studies. This will be initially by sequencing of just the coding regions of the genome (exome sequencing), but will eventually become whole genome sequencing. This new technology will progressively replace current methods of analysis for Mendelian disease, including GWA studies of complex human disease, and also gene expression microarrays. Given the declining cost of next generation DNA and RNA sequencing, and the increased density of data gained from these new methods, future discovery in human genetics is likely to accelerate. If there is enough personal health benefit from having a personal genome sequence, then we can expect that many more people will

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be analysed, and not necessarily just those with either mild and severe genetic conditions. It is likely that as more disorders are studied, that new roles for inhibins and activins may be identified, and will add to our knowledge of how these important regulatory molecules function. As many investigations will be developed without any prior knowledge of potential candidate genes, that is, a non-hypothesis based approach, it is likely that inhibins and activins may become associated with diseases that they have not been linked to previously. This will provide challenges for researchers, to determine how these genes may be involved in a variety of novel disease pathways. Therefore, it is likely that inhibins and activins will be shown to play more important roles in a range of human genetic diseases in the future. Acknowledgments I would like to thank the many collaborators, students and postdoctoral fellows over the years who have contributed to my research, especially on the role of inhibin in the development of premature ovarian failure. A special thanks to Dr. Ashwini Chand and Dr. Anna Ramachandran, whose research I have drawn on heavily to write this review. References Akutsu, K. et al., 2007. Phenotypic heterogeneity of Marfan-like connective tissue disorders associated with mutations in the transforming growth factor-beta receptor genes. Circ. J. 71, 1305–1309. Balanathan, P. et al., 2004. Epigenetic regulation of inhibin a-subunit gene in prostate cancer cell lines. J. Mol. Endocrinol. 32, 55–67. Barton, D.E. et al., 1989. Mapping of genes for inhibin subunits alpha, beta A, and beta B on human and mouse chromosomes and studies of jsd mice. Genomics 5, 91–99. Bilandzic, M., Stenvers, K.L., 2011. Betaglycan: a multifunctional accessory. Mol. Cell. Endrocrinol. 339 (1–2), 180–189. Bernard, D.J., Chapman, S.C., Woodruff, T.K., 2001. Mechanisms of inhibin signal transduction. Recent Prog. Horm. Res. 56, 417–450. Burger, H.G., Fuller, P.J., 1996. The inhibin/activin family and ovarian cancer. Trends Endocrinol. Metab. 7 (6), 197–202. Chand, A.L. et al., 2006. Mutational analysis of GDF9 and BMP15 as candidate genes in premature ovarian failure. Fertil. Steril. 86 (4), 1009–1012. Chand, A.L. et al., 2007. Functional analysis of the human inhibin A257T mutation and its potential role in premature ovarian failure. Hum. Reprod. 22 (12), 3241– 3248. Chand, A.L., Harrison, C.A., Shelling, A.N., 2010. Inhibin and premature ovarian failure. Mol. Hum. Reprod. 16 (1), 39–50. Ciarmela, P. et al., 2005. Mutational analysis of the inhibin alpha gene in preeclamptic women. J. Endocrinol. Invest. 28, 30–33. Corre, T. et al., 2009. A large-scale association study to assess the impact of known variants of the human INHA gene on premature ovarian failure. Hum. Reprod. 24 (8), 2023–2028. Coulam, C.B., Adamson, S.C., Annegers, J.F., 1986. Incidence of premature ovarian failure. Obstet. Gynecol. 67, 604–606. de Caestecker, M.P., Piek, E., Roberts, A.B., 2000. Role of transforming growth factorbeta signaling in cancer. J. Natl. Cancer Inst. 92 (17), 1388–1402. Depasquale, S. et al., 2002. Molecular analysis of inhibin A and activin A subunit gene loci in epithelial ovarian cancer. Int. J. Gynecol. Cancer 12, 443–447. Di Pasquale, E., Beck-Peccoz, P., Persani, L., 2004. Hypergonadotropic ovarian failure associated with an inherited mutation of human bone morphogenetic protein15 (BMP15) gene. Am. J. Hum. Genet. 75 (1), 106–111. Dixit, H., Deendayal, M., Singh, L., 2004. Mutational analysis of the mature peptide region of inhibin genes in Indian women with ovarian failure. Hum. Reprod. 19, 1760–1764. Dixit, H. et al., 2006. Expansion of the germline analysis for the INHA gene in Indian women with ovarian failure. Hum. Reprod. 21, 1643–1644. Findlay, J.K. et al., 2002. Recruitment and development of the follicle; the roles of the transforming growth factor-beta superfamily. Mol. Cell. Endocrinol. 191 (1), 35–43. Francis-Thickpenny, K.M. et al., 2001. Analysis of the TGF beta functional pathway in epithelial ovarian carcinoma. Br. J. Cancer 85 (5), 687–691. Frazer, K.A. et al., 2009. Human genetic variation and its contribution to complex traits. Nat. Rev. Genet. 10, 241–251. Fuller, P.J., Chu, S., 2004. Signalling pathways in the molecular pathogenesis of ovarian granulosa cell tumours. Trends Endocrinol. Metab. 15 (3), 122–128. Gatza, C.E., Oh, S.Y., Blobe, G.C., 2010. Roles for the type III TGF-beta receptor in human cancer. Cell. Signal. 22, 1163–1174.

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