Leiomyomata

Uterine leiomyomas are monoclonal, diploid, somatic cell tumors that arise from uterine smooth-muscle tissue. Leiomyomata (fibroids) are one of the most common diseases in gynecological practice, occurring in at least half of women. Although many are asymptomatic, some women manifest heavy uterine bleeding and pelvic pain resulting in hysterectomy. The inheritance pattern of fibroids is largely unknown except for some rare Mendelian types, which develop in unusual locations and are associated with other anomalies. Hereditary leiomyomatosis and renal tumors are caused by autosomal dominant mutations in the fumarate hydratase ( FH ) . Diffuse leiomyomatosis and Alport syndrome, an X-linked dominant contiguous gene deletion syndrome affecting COL4A 5 and COL4A6 , consist of glomerulonephritis, hearing loss, and eye disease .

Classical cytogenetic analysis of uterine leiomyoma demonstrates low-frequency chromosomal aberrations, including 7q deletions, trisomy 12, and translocations at chromosome 12q14, which includes the high-mobility group AT-hook 2 (HMGA2). The most significant genetic finding in uterine leiomyomata is the presence of heterozygous, clonal, somatic mutations in the mediator complex subunit 12 ( MED12 ). In their initial report, investigators found that 75% of women and 70% of uterine fibroids had variants in the MED12 gene. Half of the variants were found in exon 2, and these were predicted to be deleterious . MED12 consists of 26 subunit transcriptional regulators and is localized on chromosome Xq13.1. It is interesting that germline mutations in this gene result in X-linked intellectual disability in males without predisposition to fibroids .

Although somatic variants have been identified, it is not known if germline gene mutations contribute to the genesis and inheritance of fibroids. Several investigative methods have been employed but principally consist of genome wide association studies (GWAS), which are case-control studies to determine if certain markers (usually single nucleotide polymorphisms or SNPS) are more frequent in cases vs. controls. GWAS studies consist of two parts: the initial GWAS and a replication study. Because of the large number of loci studied, many of the findings in the initial GWAS could be found to be associated with the disorder in question by chance alone, so a replication study is required. For the replication, the markers deemed significant from the initial phase are studied in a new sample of cases and controls. Several chromosomal loci have been identified from the available GWAS in women with leiomyomata ( Table 1 ) . One study combined genome wide linkage analysis (study of family members) and GWAS. Interestingly, one SNP at 17q25.3 was in linkage disequilibrium with three genes, one of which ( FASN ), was increased threefold in leiomyoma vs. adjacent normal myometrial tissue. FASN transcripts and the FAS protein have been found to be upregulated in a variety of tumors . However, FASN association was not confirmed on subsequent replication . To date, GWAS studies provide preliminary information concerning potential genetic loci in fibroids but have not identified a gene as being causative.

Endometriosis

Endometriosis is a common hormone-dependent disorder in females resulting in pelvic pain, dysmenorrhea, and infertility. Histologically, lesions are characterized by ectopic endometrial glands and stroma located outside the uterus—commonly to other organs in the pelvis and beyond. Endometriosis is a disease of inflammatory origin, probably polygenic or multifactorial .

Similar to leiomyomata, the study of endometriosis has been complicated, and currently, no clear germline mutation has been identified. Gogusev and colleagues reported copy number variants (CNVs) in three chromosomal regions (1p36, 7p22.1, and 22q12) in patients with endometriosis . In addition, linkage analysis conducted in Australia and the UK identified significant linkage at chromosome 10q26, but no causal gene was found A GWAS study in Australia implicated loci 7p15.2 (near the HOXA10 and NFE2L3 genes) and 1p36 (containing the WNT4 gene). A Japanese GWAS also identified the locus 1p36 ( Table 1 ).

More recently, retinoid deficiency has been suggested to have a causative role in the etiology of endometriosis. Abnormal methylation of the promoters of genes such as GATA6 , ESR2 , and NR5A1 in endometrial implants leads to an increase in local estrogen and prostaglandin levels, causing the inhibition of progesterone receptors. This in turn results in the reduced synthesis and absorption of retinoids. These molecular abnormalities have detrimental effects upon cell differentiation, increased survival, and enhanced inflammation, and consequentially could result in the development of endometriosis .

PCOS

Polycystic ovary syndrome (PCOS) is one of the most prevalent endocrine disorders in females . PCOS patients demonstrate a spectrum of clinical symptoms, including hyperandrogenism, menstrual dysfunction/oligo-anovulation, and polycystic ovarian morphology . Women with PCOS are at a higher risk for infertility, dysfunctional uterine bleeding, metabolic disorders (dyslipidemia, obesity, and type 2 diabetes mellitus), and cardiovascular disease .

PCOS is a complex polygenic, multifactorial disease. Since insulin resistance and secondary hyperinsulinemia are a common finding in up to 85% of women with PCOS, insulin ( INS ) and its receptor ( INSR ) became prime candidate genes. It has been well known that mutations in INSR lead to insulin resistance, acanthosis nigricans, and type 2 diabetes mellitus. INSR mutations may also cause the more severe phenotype of the Donohue syndrome (leprechaunism), a serious autosomal recessive disease, characterized by the development of somatic abnormalities, hyperinsulinemia, hypoglycemia, hypertrichosis, and acanthosis nigricans . The molecular basis is also known for a number of congenital generalized lipodystrophies, which may overlap in phenotype with that of PCOS . Women with forms of congenital generalized lipodystrophies have a selective regional loss of body fat and extreme insulin resistance, and their phenotype may be either congenital generalized lipodystrophy (autosomal recessive) or familial partial lipodystrophy (autosomal dominant) .

Numerous candidate genes have been studied to try to understand the molecular basis of PCOS. Approaches have largely consisted of association studies, either using a candidate gene approach, transmission disequilibrium test (family based association), or GWAS ( Table 1 ). Throughout the past decade, select candidate genes have come in and out of favor as additional studies have been performed. Genes studied have included INSR FBN3 , but more recently DENND1A , FSHR , LHR, RAB5B , and THADA have been in favor. More recently, ERBB4, C9orf3 , FSHB , YAP1 , and HMGA2 have been confirmed in replication cohorts of GWAS .

Nevertheless, none of the variants identified so far is known to directly affect function. However, some of the loci identified do appear to be linked to the underlying biology of PCOS. For example, a number of the loci identified are proximate to genes that modulate or determine gonadotropic function . Further, in 2014, McAllister and colleagues also published results on the function of the product of a locus identified in PCOS GWASs . DENND1A encodes a protein associated with clathrin-coated pits, where cell-surface receptors reside. DENND1A was found in the cytoplasm and nuclei of ovarian theca cells, suggesting a role in gene regulation; immunostaining was more intense in cells from individuals with PCOS than from unaffected controls. Overexpression of DENND1A variant 2 in normal theca cells resulted in a PCOS-like phenotype, and knock-down of DENND1A.V2 in PCOS theca cells reversed this phenotype.

We should note that while the heritability of PCOS estimated in a monozygotic twin study is approximately 70% , the proportion of heritability accounted for by the PCOS loci identified so far by GWAS is <10%, although similar to that observed with other complex genetic traits. However, additional GWAS may still identify additional loci, and fine mapping of known loci might detect specific genes and functional variants of interest. In addition, other factors accounting for the high heritability of PCOS must also be investigated, including genomic structural variations and epigenetic factors. In addition, the impact of the different PCOS phenotypes (Phenotypes A and B or “classic PCOS,” Phenotype C or “ovulatory PCOS,” and Phenotype D or “non-hyperandrogenic PCOS”) on these findings remains to be determined.

Spontaneous ovarian hyperstimulation syndrome

Typically, OHSS occurs because of exogenous gonadotropins with infertility treatment. Clinical manifestations of OHSS range from mild (elevated serum estradiol levels and increased ovarian volume with abdominal pain only) to severe (the aforementioned in combination with ascites, hydrothorax, and renal and hepatic insufficiency). Interestingly, a handful of patients developed spontaneous OHSS (sOHSS) during pregnancy following natural conception. At least two familial and three sporadic cases have been described, all of whom have activating mutations of the follicle stimulating hormone receptor ( FSHR ) gene . This trait appears to be inherited in an autosomal dominant fashion, and the phenotype in affected patients consists of OHSS symptoms in the first trimester (nausea, pain, enlarged ovaries, and ascites) which resolve when the pregnancy is completed. The outcome may be live birth or miscarriage, and not every pregnancy is affected. In all cases, the mutant FSHR was able to be stimulated by human chorionic gonadotropin, which is most elevated in the first trimester, thereby explaining the onset of symptoms. In some cases, the FSHR is constitutively active (requiring no activation by FSH ligand), and in others, the mutant receptor is activated by thyroid stimulating hormone .

Mayer–Rokitansky–Kuster–Hauser syndrome

Mayer–Rokitansky–Kuster–Hauser (MRKH) syndrome, also referred to as mullerian aplasia (patients prefer the name MRKH), is a severe anomaly of the female reproductive tract. The phenotype is manifested by the absence of the vagina, cervix, and most of the uterus in women with a 46,XX karyotype. This syndrome occurs in approximately 1:5000 women and affects 10% of patients with primary amenorrhea .

Typically, MRKH syndrome has two subtypes : subtype 1, which is characterized by isolated mullerian aplasia and subtype 2, which includes additional anomalies (renal agenesis, skeletal abnormalities, congenital heart defects, and deafness) . Generally, MRKH is sporadic, but familial cases suggest an autosomal dominant pattern of inheritance with incomplete penetrance and variable expressivity or polygenic inheritance in some cases .

Numerous candidate genes ( AMH, AMHR, WT1, CFTR, WNT7A, GALT, HOXA7, PBX1, HOXA13, PAX2, HOXA10, RARG, RXRA, CTNNB1, LAMC1, DLGH1, and SHOX ) were studied in small numbers of patients, but no mutations were found. For other genes ( RBM8A, WNT9B, LHX1 , and TBX6 ), variants have been found; but their significance is unknown. Only WNT4 and HNF1B have been identified as causative genes . One family with renal cysts, maturity onset diabetes of the young (MODY) type 5, and mullerian aplasia had a heterozygous intragenic deletion of HNF1B , impairing function in vitro . The prevalence of mutations in WNT4 and HNF1B in patients with MRKH syndrome appears to be very low.

Chromosomal microarrays have been used to determine if CNVs not detectable by karyotype could be present, with many chromosomal regions implicated. Some CNVs that have been repetitively identified include ∼1 Mb deletions of 17q12 and 0.5 Mb deletions of 16p11 . Interestingly, the 17q12 CNV contains the loci for LHX1 and HNF1B , while the 16p11 CNV contains TBX6 . Despite these studies, there is no conclusive evidence that these CNVs are causative, and little else is known concerning the genetics of MRKH.

Steroid hormone resistance syndromes

Complete androgen insensitivity (CAIS) is an X-linked recessive condition that results in the absence of androgen effect in 46,XY males, which is caused by inactivating mutations in androgen receptor (AR) gene . Patients with CAIS are phenotypic women, but they lack a uterus and vagina, and have little or no axillary and/or pubic hair. Serum testosterone levels in these patients are in the normal male range. Gonadectomy should be strongly considered once pubertal development is complete since tumors more commonly occur after that time.

Although CAIS is relatively common, alternatively patients with estrogen insensitivity syndrome have only rarely been identified. The first estrogen receptor-alpha ( ESR1 ) gene mutation was identified in a 46,XY male in 1994 . The first female with an ESR1 mutation was only recently described. Notably, the patient presented with absent breast development, enlarged cystic ovaries, mild acne, and reduced bone age with significantly elevated serum estradiol levels and mildly elevated gonadotropin levels . Her ESR1 mutation significantly impaired estrogen function. Estrogen resistance is inherited as an autosomal recessive trait. Recently, another family with two affected females and an affected male were found to have ESR1 mutations with similar phenotypes .

Leiomyomata

Uterine leiomyomas are monoclonal, diploid, somatic cell tumors that arise from uterine smooth-muscle tissue. Leiomyomata (fibroids) are one of the most common diseases in gynecological practice, occurring in at least half of women. Although many are asymptomatic, some women manifest heavy uterine bleeding and pelvic pain resulting in hysterectomy. The inheritance pattern of fibroids is largely unknown except for some rare Mendelian types, which develop in unusual locations and are associated with other anomalies. Hereditary leiomyomatosis and renal tumors are caused by autosomal dominant mutations in the fumarate hydratase ( FH ) . Diffuse leiomyomatosis and Alport syndrome, an X-linked dominant contiguous gene deletion syndrome affecting COL4A 5 and COL4A6 , consist of glomerulonephritis, hearing loss, and eye disease .

Classical cytogenetic analysis of uterine leiomyoma demonstrates low-frequency chromosomal aberrations, including 7q deletions, trisomy 12, and translocations at chromosome 12q14, which includes the high-mobility group AT-hook 2 (HMGA2). The most significant genetic finding in uterine leiomyomata is the presence of heterozygous, clonal, somatic mutations in the mediator complex subunit 12 ( MED12 ). In their initial report, investigators found that 75% of women and 70% of uterine fibroids had variants in the MED12 gene. Half of the variants were found in exon 2, and these were predicted to be deleterious . MED12 consists of 26 subunit transcriptional regulators and is localized on chromosome Xq13.1. It is interesting that germline mutations in this gene result in X-linked intellectual disability in males without predisposition to fibroids .

Although somatic variants have been identified, it is not known if germline gene mutations contribute to the genesis and inheritance of fibroids. Several investigative methods have been employed but principally consist of genome wide association studies (GWAS), which are case-control studies to determine if certain markers (usually single nucleotide polymorphisms or SNPS) are more frequent in cases vs. controls. GWAS studies consist of two parts: the initial GWAS and a replication study. Because of the large number of loci studied, many of the findings in the initial GWAS could be found to be associated with the disorder in question by chance alone, so a replication study is required. For the replication, the markers deemed significant from the initial phase are studied in a new sample of cases and controls. Several chromosomal loci have been identified from the available GWAS in women with leiomyomata ( Table 1 ) . One study combined genome wide linkage analysis (study of family members) and GWAS. Interestingly, one SNP at 17q25.3 was in linkage disequilibrium with three genes, one of which ( FASN ), was increased threefold in leiomyoma vs. adjacent normal myometrial tissue. FASN transcripts and the FAS protein have been found to be upregulated in a variety of tumors . However, FASN association was not confirmed on subsequent replication . To date, GWAS studies provide preliminary information concerning potential genetic loci in fibroids but have not identified a gene as being causative.

Endometriosis

Endometriosis is a common hormone-dependent disorder in females resulting in pelvic pain, dysmenorrhea, and infertility. Histologically, lesions are characterized by ectopic endometrial glands and stroma located outside the uterus—commonly to other organs in the pelvis and beyond. Endometriosis is a disease of inflammatory origin, probably polygenic or multifactorial .

Similar to leiomyomata, the study of endometriosis has been complicated, and currently, no clear germline mutation has been identified. Gogusev and colleagues reported copy number variants (CNVs) in three chromosomal regions (1p36, 7p22.1, and 22q12) in patients with endometriosis . In addition, linkage analysis conducted in Australia and the UK identified significant linkage at chromosome 10q26, but no causal gene was found A GWAS study in Australia implicated loci 7p15.2 (near the HOXA10 and NFE2L3 genes) and 1p36 (containing the WNT4 gene). A Japanese GWAS also identified the locus 1p36 ( Table 1 ).

More recently, retinoid deficiency has been suggested to have a causative role in the etiology of endometriosis. Abnormal methylation of the promoters of genes such as GATA6 , ESR2 , and NR5A1 in endometrial implants leads to an increase in local estrogen and prostaglandin levels, causing the inhibition of progesterone receptors. This in turn results in the reduced synthesis and absorption of retinoids. These molecular abnormalities have detrimental effects upon cell differentiation, increased survival, and enhanced inflammation, and consequentially could result in the development of endometriosis .

PCOS

Polycystic ovary syndrome (PCOS) is one of the most prevalent endocrine disorders in females . PCOS patients demonstrate a spectrum of clinical symptoms, including hyperandrogenism, menstrual dysfunction/oligo-anovulation, and polycystic ovarian morphology . Women with PCOS are at a higher risk for infertility, dysfunctional uterine bleeding, metabolic disorders (dyslipidemia, obesity, and type 2 diabetes mellitus), and cardiovascular disease .

PCOS is a complex polygenic, multifactorial disease. Since insulin resistance and secondary hyperinsulinemia are a common finding in up to 85% of women with PCOS, insulin ( INS ) and its receptor ( INSR ) became prime candidate genes. It has been well known that mutations in INSR lead to insulin resistance, acanthosis nigricans, and type 2 diabetes mellitus. INSR mutations may also cause the more severe phenotype of the Donohue syndrome (leprechaunism), a serious autosomal recessive disease, characterized by the development of somatic abnormalities, hyperinsulinemia, hypoglycemia, hypertrichosis, and acanthosis nigricans . The molecular basis is also known for a number of congenital generalized lipodystrophies, which may overlap in phenotype with that of PCOS . Women with forms of congenital generalized lipodystrophies have a selective regional loss of body fat and extreme insulin resistance, and their phenotype may be either congenital generalized lipodystrophy (autosomal recessive) or familial partial lipodystrophy (autosomal dominant) .

Numerous candidate genes have been studied to try to understand the molecular basis of PCOS. Approaches have largely consisted of association studies, either using a candidate gene approach, transmission disequilibrium test (family based association), or GWAS ( Table 1 ). Throughout the past decade, select candidate genes have come in and out of favor as additional studies have been performed. Genes studied have included INSR FBN3 , but more recently DENND1A , FSHR , LHR, RAB5B , and THADA have been in favor. More recently, ERBB4, C9orf3 , FSHB , YAP1 , and HMGA2 have been confirmed in replication cohorts of GWAS .

Nevertheless, none of the variants identified so far is known to directly affect function. However, some of the loci identified do appear to be linked to the underlying biology of PCOS. For example, a number of the loci identified are proximate to genes that modulate or determine gonadotropic function . Further, in 2014, McAllister and colleagues also published results on the function of the product of a locus identified in PCOS GWASs . DENND1A encodes a protein associated with clathrin-coated pits, where cell-surface receptors reside. DENND1A was found in the cytoplasm and nuclei of ovarian theca cells, suggesting a role in gene regulation; immunostaining was more intense in cells from individuals with PCOS than from unaffected controls. Overexpression of DENND1A variant 2 in normal theca cells resulted in a PCOS-like phenotype, and knock-down of DENND1A.V2 in PCOS theca cells reversed this phenotype.

We should note that while the heritability of PCOS estimated in a monozygotic twin study is approximately 70% , the proportion of heritability accounted for by the PCOS loci identified so far by GWAS is <10%, although similar to that observed with other complex genetic traits. However, additional GWAS may still identify additional loci, and fine mapping of known loci might detect specific genes and functional variants of interest. In addition, other factors accounting for the high heritability of PCOS must also be investigated, including genomic structural variations and epigenetic factors. In addition, the impact of the different PCOS phenotypes (Phenotypes A and B or “classic PCOS,” Phenotype C or “ovulatory PCOS,” and Phenotype D or “non-hyperandrogenic PCOS”) on these findings remains to be determined.

Spontaneous ovarian hyperstimulation syndrome

Typically, OHSS occurs because of exogenous gonadotropins with infertility treatment. Clinical manifestations of OHSS range from mild (elevated serum estradiol levels and increased ovarian volume with abdominal pain only) to severe (the aforementioned in combination with ascites, hydrothorax, and renal and hepatic insufficiency). Interestingly, a handful of patients developed spontaneous OHSS (sOHSS) during pregnancy following natural conception. At least two familial and three sporadic cases have been described, all of whom have activating mutations of the follicle stimulating hormone receptor ( FSHR ) gene . This trait appears to be inherited in an autosomal dominant fashion, and the phenotype in affected patients consists of OHSS symptoms in the first trimester (nausea, pain, enlarged ovaries, and ascites) which resolve when the pregnancy is completed. The outcome may be live birth or miscarriage, and not every pregnancy is affected. In all cases, the mutant FSHR was able to be stimulated by human chorionic gonadotropin, which is most elevated in the first trimester, thereby explaining the onset of symptoms. In some cases, the FSHR is constitutively active (requiring no activation by FSH ligand), and in others, the mutant receptor is activated by thyroid stimulating hormone .

Mayer–Rokitansky–Kuster–Hauser syndrome

Mayer–Rokitansky–Kuster–Hauser (MRKH) syndrome, also referred to as mullerian aplasia (patients prefer the name MRKH), is a severe anomaly of the female reproductive tract. The phenotype is manifested by the absence of the vagina, cervix, and most of the uterus in women with a 46,XX karyotype. This syndrome occurs in approximately 1:5000 women and affects 10% of patients with primary amenorrhea .

Typically, MRKH syndrome has two subtypes : subtype 1, which is characterized by isolated mullerian aplasia and subtype 2, which includes additional anomalies (renal agenesis, skeletal abnormalities, congenital heart defects, and deafness) . Generally, MRKH is sporadic, but familial cases suggest an autosomal dominant pattern of inheritance with incomplete penetrance and variable expressivity or polygenic inheritance in some cases .

Numerous candidate genes ( AMH, AMHR, WT1, CFTR, WNT7A, GALT, HOXA7, PBX1, HOXA13, PAX2, HOXA10, RARG, RXRA, CTNNB1, LAMC1, DLGH1, and SHOX ) were studied in small numbers of patients, but no mutations were found. For other genes ( RBM8A, WNT9B, LHX1 , and TBX6 ), variants have been found; but their significance is unknown. Only WNT4 and HNF1B have been identified as causative genes . One family with renal cysts, maturity onset diabetes of the young (MODY) type 5, and mullerian aplasia had a heterozygous intragenic deletion of HNF1B , impairing function in vitro . The prevalence of mutations in WNT4 and HNF1B in patients with MRKH syndrome appears to be very low.

Chromosomal microarrays have been used to determine if CNVs not detectable by karyotype could be present, with many chromosomal regions implicated. Some CNVs that have been repetitively identified include ∼1 Mb deletions of 17q12 and 0.5 Mb deletions of 16p11 . Interestingly, the 17q12 CNV contains the loci for LHX1 and HNF1B , while the 16p11 CNV contains TBX6 . Despite these studies, there is no conclusive evidence that these CNVs are causative, and little else is known concerning the genetics of MRKH.

Steroid hormone resistance syndromes

Complete androgen insensitivity (CAIS) is an X-linked recessive condition that results in the absence of androgen effect in 46,XY males, which is caused by inactivating mutations in androgen receptor (AR) gene . Patients with CAIS are phenotypic women, but they lack a uterus and vagina, and have little or no axillary and/or pubic hair. Serum testosterone levels in these patients are in the normal male range. Gonadectomy should be strongly considered once pubertal development is complete since tumors more commonly occur after that time.

Although CAIS is relatively common, alternatively patients with estrogen insensitivity syndrome have only rarely been identified. The first estrogen receptor-alpha ( ESR1 ) gene mutation was identified in a 46,XY male in 1994 . The first female with an ESR1 mutation was only recently described. Notably, the patient presented with absent breast development, enlarged cystic ovaries, mild acne, and reduced bone age with significantly elevated serum estradiol levels and mildly elevated gonadotropin levels . Her ESR1 mutation significantly impaired estrogen function. Estrogen resistance is inherited as an autosomal recessive trait. Recently, another family with two affected females and an affected male were found to have ESR1 mutations with similar phenotypes .