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Endocrine Reviews 23 (3): 382-392
Copyright © 2002 by The Endocrine Society

Genetic Approaches to Unraveling Reproductive Disorders: Examples of Bedside to Bench Research in the Genomic Era

Stephanie B. Seminara and William F. Crowley, Jr.

Reproductive Endocrine Unit and Harvard-Wide Reproductive Endocrine Sciences Center and National Center for Infertility Research, Massachusetts General Hospital, Boston, Massachusetts 02114

Correspondence: Address all correspondence and requests for reprints to: Stephanie Seminara, M.D., Reproductive Endocrine Unit, Bartlett Hall Extension 505, Fruit Street, Boston, Massachusetts 02114. E-mail: seminara.stephanie{at}mgh.harvard.edu


   Abstract
Top
 Abstract
I. Introduction
 II. Commonly Used Genetic...
 III. Use of Unusual...
 IV. Use of Linkage...
 V. Polycystic Ovary Syndrome...
 VI. Conclusions
 References
 
Despite the rapid advances in medical genetics, many clinicians and investigators remain unaware of the general approaches that can be used to map genes. Although there are specific challenges to using genetic approaches in reproductive medicine, the following report summarizes mapping efforts for three diseases: adrenal hypoplasia congenita, hypergonadotropic ovarian failure, and polycystic ovary syndrome. The themes of rare and novel phenotypes, genetically homogenous populations, and genotype/phenotype correlations are emphasized.

I. Introduction

II. Commonly Used Genetic Approaches

A. The importance of DNA variants

B. Linkage analysis

C. Association studies

III. Use of Unusual Phenotypes: DAX1

IV. Use of Linkage Analysis to Determine a Cause of Hypergonadotropic Ovarian Failure

V. Polycystic Ovary Syndrome (PCOS) and Association Approaches

VI. Conclusions


   I. Introduction
Top
 Abstract
 I. Introduction
II. Commonly Used Genetic...
 III. Use of Unusual...
 IV. Use of Linkage...
 V. Polycystic Ovary Syndrome...
 VI. Conclusions
 References
 
OVER THE PAST two decades, medical genetics has moved from being a tool of the basic investigator into the mainstream of medical practice. Identification of the hereditary contributions to common endocrine disorders, illuminations of molecular pathophysiology of known conditions, development of new predictive tests for genetic abnormalities, and applications in the field of pharmacogenomics are all new topics that now regularly appear in our journals. For those considering careers in investigation, as well as for established researchers, the meteoric progress of the Human Genome Project has left a considerable educational gap in its wake. One of these current lacunae is a working knowledge of the general approaches that can be used by clinical investigators in collaboration with thoughtful clinicians to map new genes using their relatively unique patient populations. Thus, patients can generate remarkable new biological opportunities for both basic and clinical scientists. This evolving trend of "bedside to bench" research is the focus of this overview and one well suited to endocrinologists for several reasons. As a society, our practitioners, clinical investigators, and basic researchers still interact at a single annual meeting that provides both a forum and a tradition for such collegial dialogues to occur. As investigators, both clinical and basic scientists are faced with similar challenges, incorporating the new information and approaches of the human genome into the fabric of their daily existence as quickly as possible. Perhaps most importantly, our specialty cherishes these collegial interactions as a mechanism by which to improve the plight of patients with endocrine disorders. This report focuses on the approaches currently being employed to map genes in human reproduction, which is a particularly difficult field in which to use genetics. Reproductive defects limit family sizes, in many ways acting like lethal diseases. Thus, they often result in small family sizes, rendering certain genetics approaches ineffective. Despite this difficulty, such approaches have made major contributions to our understanding of several disorders. Several common themes emerge from their use, including the importance of: 1) rare and novel phenotypes, 2) genetically homogenous populations with heritable disorders, 3) major advances from basic science that continually bring forth new candidate genes, and 4) understanding genotype/phenotype correlations. These are all critical components of successful mapping efforts, and ones that rely upon clinicians and clinical investigators being familiar with these approaches.


   II. Commonly Used Genetic Approaches
Top
 Abstract
 I. Introduction
 II. Commonly Used Genetic...
III. Use of Unusual...
 IV. Use of Linkage...
 V. Polycystic Ovary Syndrome...
 VI. Conclusions
 References
 
A. The importance of DNA variants
Fundamental to any understanding of the use of the human genome to discover disease genes is an appreciation of common variations in DNA sequences and their utility in localizing "addresses" of genes within the human genome. Most disease-mapping efforts depend critically on the discovery that variations in human genetic sequences occur frequently in the human genome. These normally occurring variations can sometimes alter the function of the gene in which they occur by either increasing or decreasing its activity to varying degrees. When detrimental, such variants are often rare. Other variants may not change the function of a gene dramatically but, when inherited in tandem with other equally mild, but synergistic, genetic variants, may confer a susceptibility to human disease. These genetic variants may occur more frequently in certain populations because there is no individual selection pressure against their transmission. Since the identification of the first molecular polymorphisms in the ABO blood group at the beginning of the 20th century (1), there has been a continuous evolution in the discovery and detection of DNA variants. One of the first genetic markers to be used was the RFLP (restriction fragment length polymorphism), caused by base substitutions in DNA that create or destroy specific restriction endonuclease cleavage sites (2). Each restriction enzyme recognizes specific DNA sequences of approximately 4–8 bp. When DNA from a patient with a RFLP is cut with a restriction enzyme, the typical banding pattern of DNA is altered from that occurring in normals. It is important to emphasize that the nucleotide variation creating the RFLP itself may not cause any clinical or biochemical change in the individual. Rather, the presence of a specific RFLP can be used as a genetic marker to trace the segregation of DNA and, by extension, the inheritance of a disease allele within a family. Higher levels of heterozygosity are associated with a different type of variant or polymorphism known as the VNTR (variable number of tandem repeats, or "minisatellites") (3). VNTRs, which also occur frequently, consist of longer repeated nucleotide sequences of approximately 10–60 bp. Microsatellites (as opposed to these minisatellites) are yet a third class of polymorphic markers in which the repetitive sequences are composed of smaller units of di-, tri-, or tetra-nucleotide repeats (4). Microsatellite repeats often consist of the nucleotides cytosine and adenosine. When the number of repeats within a microsatellite differs between individuals, the marker is said to be polymorphic. For example, suppose that a family presents with a putative recessive disorder (offspring are affected, parents are carriers). Amplification and electrophoresis of a given marker might reveal that the father contains two alleles, one of 200 and the other of 206 bp, whereas the mother contains two other alleles, 204 and 208 bp. As each parent carries two distinct alleles for this microsatellite, this marker is said to be highly polymorphic. The particular pattern of inheritance of the marker alleles in the children could then be used to calculate the likelihood that the marker is "linked" to the disease gene (see Section II.B, below for discussion of linkage). The high frequency and ease of amplification by PCR of microsatellite markers have made them one of the most commonly used tools in genetic mapping in the past decade.

By far, the most plentiful source of DNA variation in the human genome is represented by single nucleotide polymorphisms (SNPs). SNPs are biallelic sequence variants distributed at a very high frequency throughout the genome, approximately 1 in every 1000 bp (5, 6, 7). Although each nucleotide has the theoretical possibility of four SNP alternatives, the term biallelic is often used to describe SNPs because the frequencies of the four basic types are not equal. One common SNP involves a C->T substitution as many cytosine residues are methylated and can spontaneously deaminate to yield a thymidine, particularly at CpG islands (8). Because only two alternative bases typically occur at each polymorphic position, SNPs are less informative than microsatellite markers. However, SNPs are much more common (5). The development of rapid throughput DNA microarrays has advanced the ability to identify and catalog such plentiful DNA variation. Although there are numerous uses of microarrays (i.e., gene expression profiling), the most straightforward application of this technology is analysis of DNA variation. Given a region of DNA, four probes are designed to "interrogate" a single pair position. One probe is a perfect complement; the other three substitute one of the other base pairs at the interrogation position (9). If a substitution variant is present, the probe containing the complementary variant will provide the greatest signal. Although not well suited to polymorphic repeats, deletions, or insertions, the variation identified by use of these SNP chips can be applied to linkage, linkage disequilibrium, and loss of heterozygosity studies (see below). Therefore, although each SNP may be less informative than a traditional minisatellite, the development of these high-throughput genotyping capabilities can compensate for the large numbers of SNPs needed in contemporary study designs.

B. Linkage analysis
Linkage analysis is used to demonstrate the cosegregation of a genetic variant and a disease locus. The genetic variants used can be any of the ones described above, including RFLPs, VNTRs, or microsatellites, although the latter have been the workhorse tool of recent years. The pattern of variation for a particular marker is determined in a pedigree and juxtaposed against the presence or absence of the disease phenotype. The data obtained from each marker are then used to generate a LOD score. The LOD score is simply the log10 of a ratio, with the numerator being the odds that the marker and the disease locus are linked divided by the odds that the loci are unlinked. Because the actual distance between a marker and the disease locus is unknown, the LOD score is traditionally calculated for fixed recombination fractions ({theta}). The greater the recombinant events observed for a given marker, the greater the distance between the marker and the disease locus. For simple Mendelian traits, LOD score calculations require precise inheritance models, knowledge of disease gene penetrance, disease gene frequency, and spectrum of phenotypes. The simplest application of linkage analysis is the identification of a disease locus in a single large pedigree, although multiple pedigrees can also be analyzed together. Because the use of one or more families in a linkage study can unwittingly introduce genetic heterogeneity, great care is usually taken to choose families of sufficient size that are relatively homogenous, either by phenotype, ethnic derivation, etc. Perhaps the greatest advantage of the linkage approach is its ability to localize a disease locus without any prior knowledge or assumptions of the genes that might be involved in causing a given disease. Linkage approaches are most powerful when applied to single-gene (Mendelian) disorders and much less so when used with complex, multifactorial diseases in which the role of any single gene is modest. Despite the numerous successes of this approach, linkage analysis can still fail if: 1) the number of patients in a family is insufficient to support a rigorous statistical analysis; 2) incorrect phenotypes are assigned; 3) the phenotype is mimicked by more than one genetic locus (phenocopies); or 4) the assigned mode of inheritance is incorrect. Although linkage approaches have the powerful ability to localize a disease-causing gene to a particular chromosomal region, they do not allow a determination of which of the many genes at that particular genomic address is responsible for the disease under study. Thus, testing each individual gene or genes at a linked locus for mutations (positional cloning) can be quite arduous. However, the availability of the full human DNA sequence provided by the Human Genome Project has now greatly simplified this process. Classical linkage analysis requires that the mode of inheritance of the disease transmission is known (i.e., parametric analysis). However, additional methods of analysis have been developed in which the precise determination of the mode of inheritance occurring in a disease is not required. These nonparametric methods are based on the principle of allele sharing among affected individuals without the imposition of an inheritance model. Investigators must then demonstrate that relatives within a kindred with a given disease inherit identical copies of a particular chromosomal region more often than expected by chance. The simplest application of this method is to determine the ability of pairs of siblings affected with a disease to share a given haplotype as compared with that which might occur by chance. This is called sib-pair analysis (10) and is particularly useful if investigators have collected DNA from large numbers of families with a disease in which sufficient sibs (with and without the disease) are available for analysis at a given gene or polymorphic site.

C. Association studies
Linkage analysis and allele-sharing methods rely on the inheritance of adjacent DNA variants over generation(s). Association studies also rely on the inheritance of DNA variants but over many more generations. Therefore, whereas linkage analysis determines whether a variant/haplotype is linked to a disease locus within a pedigree or collection of pedigrees, association studies assess whether a genetic variant is linked to a disease locus on a population scale. In essence, association studies are large linkage studies but of hypothetical pedigrees (11). They are generally performed with a given candidate gene(s) in mind. A genetic marker near or within a candidate gene is "associated" with a disease if the frequency of occurrence of that marker is higher in affected individuals than unaffected individuals. Association studies are thus well suited to diseases in which the mode of inheritance is uncertain, the phenotype is heterogenous or variable in its time of onset, and/or the disease is characterized by variable penetrance. Although linkage analysis is a better method for looking at loci that are necessary for disease expression, association studies are preferable for finding "susceptibility" loci (12). These loci are often high-frequency, low-risk alleles that do not produce robust signals in family linkage studies. Despite the frequent use of association approaches, many published associations are often not corroborated in subsequent studies (13). A false-positive association can occur if the allele under study does not actually cause the disease but is in linkage disequilibrium with an allele that does. This circumstance arises when the allele under study and the trait-causing allele have not been separated by the recombination of DNA that occurs over time. Because linkage disequilibrium depends on a population’s history, a disease may demonstrate false-positive associations to different alleles in various subpopulations. Another cause of a false-positive association is population admixture, which is due to the structure of the population under study rather than true linkage disequilibrium. For example, suppose one is studying whether marker A plays a role in causing disease A in population X. Also suppose that population X contains an ethnic group Y; marker A occurs more commonly in group Y and disease A occurs more frequently in group Y. Thus, an association study might conclude that a positive association exists between disease A and marker A simply because marker A is more common in subgroup Y when compared with the rest of the population X. One way to avoid false-positive associations is to use unaffected family members as controls, as opposed to unrelated individuals. This protects against the false associations caused by such population heterogeneity (14). Family-based linkage disequilibrium tests, such as the transmission-disequilibrium test (TDT) (15), determine whether parents heterozygous for a disease allele transmit that allele more often to their affected children than the nondisease allele. Originally used as a confirmatory analysis once association had been established, family-based linkage disequilibrium tests now suffice as initial, stand-alone tests. In fact, numerous family-based methods, most based on the TDT, have now been developed (16). Some extend the original TDT to multiallelic markers, multiple markers, missing parental information, and extended pedigrees. Other methods go beyond single-marker genotypes to the transmission of haplotypes extending over several adjacent markers. It is important to recognize that the methodology of association testing is rapidly evolving.


   III. Use of Unusual Phenotypes: DAX1
Top
 Abstract
 I. Introduction
 II. Commonly Used Genetic...
 III. Use of Unusual...
IV. Use of Linkage...
 V. Polycystic Ovary Syndrome...
 VI. Conclusions
 References
 
In all areas of medicine, patients with complex syndromes and/or unusual phenotypes represent unique biological opportunities for mapping disease-causing genes. Over 20 yr ago, a family was identified with 46 XY gonadal dysgenesis (phenotypic females with rudimentary streak gonads) and an X-linked mode of inheritance (17). This discovery led to the hypothesis that a gene on the X chromosome was involved in human sex determination. This hypothesis was strengthened when additional patients with sex reversal were identified, each of whom had a duplication in the Xp region (18, 19, 20, 21, 22, 23). To determine the locus responsible for male-to-female sex reversal (known as DSS for dosage-sensitive sex reversal), the extent of Xp duplication was compared in four sex-reversed men and in four men with normal testis differentiation (24). Using dosage analysis, the DSS locus was originally mapped to a region of approximately 20 megabases within Xp21.2–p22.1. Next, a submicroscopic deletion in a 46 XY sex-reversed female confined the DSS locus to a 160-kb region of Xp21, adjacent to the adrenal hypoplasia congenita (AHC) locus. AHC is a condition characterized by adrenal insufficiency and hypogonadotropic hypogonadism but with normal male external genitalia. Several patients had been identified with a complex syndrome involving AHC, glycerol kinase deficiency, and Duchenne muscular dystrophy, suggesting that these disease genes resided near one another on the X chromosome. Many 46 XY patients affected by an apparent AHC-glycerol kinase-Duchenne muscular dystrophy deletion syndrome were found to be completely deleted for the DSS critical region, suggesting that duplications of this locus can disrupt testis formation but abnormal/absent loci are compatible with a male phenotype. Using probes to delimit the proximal and distal limits of critical intervals, investigators were eventually able to clone the gene(s) underlying DSS and AHC (25). The gene that was identified, DAX1 (dosage-sensitive, sex-reversal, adrenal hypoplasia congenita, critical region on the X chromosome), encodes a 470-amino acid protein containing two exons. The C-terminal half resembles the ligand binding domain of members of the nuclear hormone receptor gene family. The novel amino terminus of DAX-1 contains three and one half repeats of a 65- to 67-amino acid motif and lacks the zinc finger motif typically present in the DNA binding domain of other nuclear hormone receptors. Loss-of-function deletions and intragenic mutations in DAX1 in patients with AHC provided final evidence that the absence of the DAX1 gene product is responsible for the combined phenotype of AHC and idiopathic hypogonadotropic hypogonadism (IHH). In subsequent studies, a range of frameshift and nonsense mutations has been reported throughout DAX1 in patients with AHC and IHH (26, 27, 28). These mutations cause truncation of the functionally important carboxy-terminal region of the protein. Missense mutations in DAX1 are relatively rare and, to date, are also localized within the putative ligand binding domain alone (29, 30). Final confirmation that DAX1 actually is the gene responsible for DSS was not produced until 1998, when overexpression of Dax1 was seen to induce sex reversal in male mice expressing a reduced functional level of another sex determining gene, sex-determining region Y gene (SRY) (31). Thus, the search for the gene for DSS came full circle from the original phenotype that drew attention to its presence. DAX1 has a complex pattern of expression during development, being present in both the fetal and adult adrenal gland (32). The protein is also expressed in the developing diencephalon and pituitary gonadotrophs in mice, and in the hypothalamus and pituitary gland in humans (32, 33). In the developing mouse gonad, expression of Dax-1 rapidly declines in the testis but is maintained in the ovary (33). This sexually dimorphic pattern of expression in development again suggests that Dax-1 plays distinct roles in sex determination, either as a repressor of testicular differentiation or as a regulator of ovarian development. Although its precise physiological role remains to be elucidated, a number of pathways point to a role for DAX-1, paradoxically, as a repressor of gene transcription. DAX-1 binds to hairpin secondary structures of DNA and blocks steroidogenesis in adrenal cells via its transcriptional repression of the steroidogenic acute regulatory protein gene (34). The C terminus of DAX-1 contains potent transcriptional silencing activity that can repress the activity of various promoters (35), and has been implicated as a potent inhibitor of SF-1, another orphan nuclear receptor with an important role in the development of the adrenal gland and the gonads (36). Finally, DAX-1 recruits the important nuclear receptor corepressor molecule, N-CoR, to steroidogenic factor 1 (37). Thus, many molecular mechanisms for DAX-1-mediated repression exist, including direct DNA binding, transcriptional silencing, and recruitment of corepressors. Although the hypogonadotropic hypogonadism associated with AHC is conventionally perceived as a congenital disorder, subsequent expanded phenotyping of patients with confirmed DAX1 mutations have recently challenged this perception. Normal gonadotropin and testosterone levels have been documented in newborn males with AHC and DAX1 mutations (38, 39, 40). Spontaneous pubertal development has also been reported in patients with DAX1 mutations, although that pubertal development is incomplete (41). Taken together, these findings suggest that the mechanisms by which DAX-1 controls the neonatal activation of the hypothalamic-pituitary-gonadal axis may be distinct from those controlling puberty or that the hypogonadotropic hypogonadism of AHC worsens over time. Hypogonadotropic hypogonadism is conventionally thought of as a treatable disorder, either using the exogenous GnRH pump to stimulate endogenous production of LH and FSH or administering gonadotropin therapy to stimulate gametogenesis. Although only limited data are available, patients with AHC/IHH again demonstrate unusual features that stretch the current understanding of the hypogonadotropic phenotype, sometimes demonstrating a response to GnRH and sometimes proving resistant to its administration (42, 43, 44, 45, 46). In addition, our group (40) treated an AHC patient for 3 yr with escalating doses of both human chorionic gonadotropin and FSH but failed to induce spermatogenesis despite an increase in testicular size, from 5 to 10 cc, and the achievement of normal testosterone levels documenting the health of his Leydig cells. Testicular biopsy in this case demonstrated curious defects in spermatogenesis that subsequently became interpretable when the mouse knockout became available. Other groups (47, 48, 49) have reported similar difficulty inducing spermatogenesis with gonadotropin therapy. These findings suggest that even when bypassing the hypothalamic-pituitary axis with exogenous gonadotropins, AHC patients can harbor an intrinsic gonadal defect in their Sertoli/germ cells.

The direct role for DAX-1 in achieving normal spermatogenesis is clearly demonstrated by studies of mice in which this gene has been deleted. Mutant male mice have normal testosterone and gonadotropin levels but are infertile, with progressive degeneration of the germinal epithelium, loss of spermatogenesis, dilation of the seminiferous tubules, and Leydig cell hyperplasia (50). Subsequent Sertoli cell-specific expression of a DAX1 transgene can restore fertility and fecundity in these knockout mice, although testicular morphology improves only modestly. These findings suggest that Dax1 expression in non-Sertoli cells is important for normal testicular development (51). Contributing to the infertility in Dax1-deficient mice is abnormal differentiation, proliferation, and/or sloughing of Leydig and Sertoli cells into the seminiferous tubular lumen that causes subsequent obstruction of the rete testis (52). Although the clinical features of X-linked diseases are traditionally thought to be expressed only by males, once again, detailed phenotyping of families with mutations in DAX1 has widened the spectrum of associated phenotypes to include females. A woman with isolated hypogonadotropic hypogonadism was found to have a homozygous DAX1 mutation from spontaneous gene conversion (53). This nonsense mutation occurred in the DNA binding domain and should have prevented the synthesis of a functional protein. Interestingly, the identical mutation was carried in a hemizygous fashion by the woman’s father, who apparently had normal fertility. Our group (40) reported an extended family with a DAX1 deletion leading to a premature stop codon in which three generations of women, all documented heterozygote carriers of the mutation, demonstrated delayed puberty with menarche occurring at ages 17–18 yr. Despite these revealing findings in patients with individual DAX1 mutations, consistent genotype/phenotype correlations have been largely elusive for AHC. Age of presentation of patients’ adrenal dysfunction appears to vary widely, even between siblings carrying the same DAX1 mutation (30). Because an individual’s genotype does not appear to be an accurate predictor of phenotype, it is likely that activity thresholds, modifying genes, and/or environmental influences may a play a role in affecting clinical presentation (54). However, there are some exceptions to this general observation. The DAX1 mutations R267P, {Delta}V269, and carboxy-terminal deletion {Delta}448–470 are associated with clinically severe phenotypes and have been shown to reduce the inhibitory activity of DAX-1 in transient gene expression assays (35, 36, 37). A patient bearing an I439S mutation demonstrated a less severe clinical phenotype with mild adrenal failure and incomplete hypogonadotropic hypogonadism occurring in early adulthood (48). This mutant had less effect on DAX-1 repressor activity than the other mutants when tested in vitro. Similarly, a patient was described with a Y380D mutation who presented at age 28 with hypogonadism and compensated adrenal failure and whose mutation caused partial loss of function in transient gene expression assays (49). Although these genotype/phenotype correlations remain the exception rather than the rule in AHC, they demonstrate that mutations in DAX1 should be considered in patients presenting with adult onset or partial hypogonadotropic hypogonadism and mild hypoadrenalism. The story of DAX1 continues to evolve rapidly through the combination of clinical investigation and molecular expression studies. The discovery of this gene was accelerated by a contemporary search for a DSS locus. After its discovery, DAX1 has added layers of complexity to the traditional understandings of hypogonadotropic hypogonadism and X-linked diseases—defects at the hypothalamus, pituitary, and gonad; failure to induce spermatogenesis with traditional therapies; phenotypes in males and females; and a paucity of clear genotype/phenotype correlations. Thus, it is an excellent example of a disorder in which gene identification techniques, clinical investigation, and coordinated molecular studies have led to a broad series of advances with importance in the development of the pituitary, adrenal, and testis.


   IV. Use of Linkage Analysis to Determine a Cause of Hypergonadotropic Ovarian Failure
Top
 Abstract
 I. Introduction
 II. Commonly Used Genetic...
 III. Use of Unusual...
 IV. Use of Linkage...
V. Polycystic Ovary Syndrome...
 VI. Conclusions
 References
 
FSH is critical for gametogenesis in many species including the human. In women, FSH plays a critical role in follicular selection and maturation, estrogen production by granulosa cells, and control of several genes whose downstream effects produce ooctye maturation. In the male, FSH initiates spermatogenesis, Sertoli cell proliferation, and seminiferous tubular differentiation. Loss-of-function mutations in the FSH receptor gene (FSHR) might therefore be expected to have more severe phenotypic consequences in a female than in a male because it is a key stimulator of aromatization of androgen precursors and, hence, estrogen production. In the male, small testes with abnormal spermatogenesis would be the anticipated phenotype. Therefore, mutation analysis of the FSHR was conducted in a variety of clinical states including premature ovarian failure (55, 56, 57), hypergonadotropic hypogonadism (58), resistant ovary syndrome (57), polycystic ovary syndrome [PCOS (57)], oligo/azoospermia and high FSH levels (59), and men with idiopathic infertility (60). However, loss-of-function mutations in FSHR were not detected in any of these conditions. Yet, the important role of the FSHR in human reproduction was uncovered by a linkage study in Finland (61). Given the genetic homogeneity of this country, it was anticipated to be a rich source of recessively inherited diseases. Very few examples of classical linkage analysis exist in reproductive medicine, due to the reproductive lethality of many disease models. However, the presence of multiplex families in Finland with hypergonadotropic ovarian dysgenesis (ODG) allowed investigators to pursue a linkage approach to determine the genetic and molecular basis of a subset of patients with this disease (61). ODG is characterized by primary amenorrhea, streak ovaries, and a normal XX karyotype. Although rare and mostly sporadic in its occurrence, a review of histologically verified cases of XX gonadal dysgenesis in Finland revealed frequent familial aggregation of affected individuals (62). A nationwide population-based study of Finnish women born over a 25-yr period uncovered 75 cases of the disease (63). In eight of these families, more than one female was affected. Genealogical studies demonstrated that most of these affected families originated from an isolated subpopulation in Finland, and segregation analysis supported the clinical suspicion of an autosomal recessive gene causing ODG in this unique genetic environment. These multiplexed families were then used to search for the genetic and molecular basis for ODG gene using polymorphic markers and linkage analysis. Thirty samples from six multiplex families were used in the initial linkage studies (61), and preliminary evidence for linkage was established on chromosome 2p; this finding was confirmed using additional markers. Data from three markers established that a common haplotype was present in all affected chromosomes, suggesting that all kindreds shared one ancestral disease-associated mutation. Recombination events established a candidate region of approximately 10.5 cM. Three factors then came into play. First, both the FSH and LH receptor genes (FSHR, LHR) had already been assigned to this candidate region on 2p (64, 65) Second, disease-causing mutations had already been identified in the transmembrane domains of the LHR (66, 67, 68, 69). Thirdly, the phenotype of the LHR mutations was different from that encountered in the Finnish families. Hence, the FSHR was screened for mutations, and all affected women were found to be homozygous for C566T, which predicts an amino acid change of Ala at position 189 to Val. When immortalized mouse Sertoli cells were transfected with the mutated FSHR, the binding affinity was similar to the wild-type receptor, but the binding capacity was markedly diminished, being only 3% of the wild-type cells (61). In addition, cAMP production was markedly reduced in response to recombinant FSH. If, however, the reduced number of FSHRs on the cell membrane were to be taken into account, the cAMP stimulation was grossly normal. Although additional studies would be needed to explore the low binding capacity with normal binding affinity (i.e., intracellular trafficking or accelerated receptor degradation), the Ala189Val substitution was the first genetic change to be characterized in detail as a cause of ODG. Twenty-two patients with the FSHR mutation were then compared with 30 women with ODG with normal FSHRs (70). Whereas both groups had postmenopausal gonadotropin levels and variable development of secondary sex characteristics, the patients with FSHR mutations had primary amenorrhea. Although the ovaries from the patients with FSHR mutations were hypoplastic, all had follicles present. All nine patients had primordial follicles, six had primary, two had preantral, and one patient had a mature follicle. This surprising finding demonstrates the FSH independence of primordial follicle recruitment and early follicular growth. In contrast, in the four cases in which the FSHR mutation was not detected, no primordial follicles and only one primary follicle were observed. Thus, the patients with FSHR mutations have ovaries that represent a distinct form of ODG. In the Finnish cohort, five homozygous males were then identified with the Ala189Val mutation (71). All were healthy and normally masculinized with normal testosterone levels, normal or slightly elevated LH levels, and moderately elevated FSH levels. All had low to normal testicular volumes and abnormal semen analyses. Four had oligospermia, and one had normal sperm concentration but low volume and teratozoospermia. Although the Ala189Val mutation in females causes follicular arrest and infertility, its suppression of spermatogenesis in men was more variable. However, before taking these findings to indicate that FSH action is not essential for spermatogenesis, it is important to recall that this mutation is not a complete knockout of function in vitro. Whereas the phenotype in females may suggest that it is severe, it may well be that FSH action is backed up or supported by other systems in the male but not in the female. In support of this notion, a number of studies (72, 73) have demonstrated FSH to be necessary for initiation of spermatogenesis at puberty, but FSH appears to be less critical in maintenance of spermatogenesis in the adult. In fact, human chorionic gonadotropin or androgens alone can reinitiate spermatogenesis in the adult male in the absence of FSH (74). The lessons learned from the discovery of the FSHR-mutation patients have expanded into multiple levels of gonadotropin biology: activating and inactivating mutations of both gonadotropins and their receptors have been identified and characterized, and this story continues to expand.


   V. Polycystic Ovary Syndrome (PCOS) and Association Approaches
Top
 Abstract
 I. Introduction
 II. Commonly Used Genetic...
 III. Use of Unusual...
 IV. Use of Linkage...
 V. Polycystic Ovary Syndrome...
VI. Conclusions
 References
 
PCOS is the most common endocrinopathy of reproductive-age women. Despite its prevalence, controversy still exists over the precise diagnostic criteria for this condition. In 1990, a conference at the NIH/National Institute of Child Health and Human Development used the following criteria for diagnosis: oligo-ovulation, hyperandrogenism on physical exam, and/or hyperandrogenemia documented on laboratory testing with exclusion of other endocrinopathies. Polycystic ovary morphology identified on ultrasound can be seen in ovulatory women with hyperandrogenemia (75) and in roughly 20% of otherwise endocrinologically normal women (76, 77). The male phenotype for PCOS has not been clearly identified. The potential metabolic consequences of PCOS are vast. The association of PCOS and hyperinsulinemia has been documented for 20 yr (78). PCOS women have marked peripheral resistance that is independent of obesity and defects in insulin secretion, suggesting that this syndrome is characterized by a unique abnormality in the insulin pathway (79). Recent studies suggest that 50% of obese PCOS women are insulin resistant when compared with appropriately age- and weight-matched controls (80). Insulin resistance is recognized as a major risk factor for the development of type 2 diabetes (81, 82). In addition, PCOS women demonstrate ß-cell dysfunction (83, 84, 85), a second risk factor for type 2 diabetes. Although the high rates of insulin resistance and glucose intolerance in PCOS (86) would suggest that PCOS women are at increased risk for type 2 diabetes, there are no prospective studies to demonstrate this. However, a long-term follow-up study of postmenopausal women with a history of PCOS revealed a 13% prevalence of type 2 diabetes compared with less than 2% in a control population (87). Although the metabolic abnormalities of PCOS and their long-term health ramifications have received most of the spotlight recently, it is the reproductive phenotype that presents several challenges for genetic investigation. These include: 1) the lack of a universally accepted diagnostic criteria with which to make a diagnosis; 2) the absence of definition of a clear male phenotype; and 3) the high prevalence of polycystic ovaries in otherwise normal women. These challenges confound the segregation analyses that have been used to determine whether there is a high incidence of PCOS in affected relatives. These studies are flawed by use of different diagnostic criteria, failure to exclude other causes of hyperandrogenism (i.e., 21-hydroxylase deficiency), and different methods of patient ascertainment (direct observation in some studies and questionnaire in others). There clearly appears to be evidence for a genetic component in PCOS, based on the familial clustering of cases. Hyperandrogenism appears to be the strongest genetically inherited characteristic in familial cases (88). In four studies (89, 90, 91, 92), segregation analysis has suggested that PCOS follows an autosomal dominant mode of inheritance. However, an additional study (93) suggested that the prevalence of PCOS morphology in the sisters of PCOS probands exceeded the expectations that would be predicted by an autosomal dominant mode of transmission. Another study (94) suggested that PCOS has an X-linked mode of inheritance. Although the majority of studies suggest autosomal dominance, the limitations of the investigative designs and the lack of clear uniformity in the results suggest that PCOS may be best suited to genetic analyses without any presumption of the mode of inheritance. In fact, PCOS appears to be similar to other complex diseases that may be caused by the interactions of several genes. Therefore, most investigators to date have taken a candidate-gene approach to studying the genetics of PCOS, using association studies and nonparametric-linkage-analysis approaches making no assumption about the mode of inheritance. Many of the candidate genes that have been selected for testing act in either the androgen-biosynthetic pathway or metabolic pathways involved in insulin action. For example, a common variant form of the gene encoding P450c17a (CYP17) that encodes 17-hydroxylase/17,20 lyase was tested for evidence of association in PCOS families. Although initial studies suggested an association (95), subsequent, larger studies failed to confirm this finding (96, 97, 98). Variation at the cholesterol side-chain cleavage gene, CYP11A, was, however, found to be positively associated with PCOS and serum testosterone levels (99). In addition, multipoint analysis demonstrated excess allele sharing at CYP11A, suggesting that this was a susceptibility locus for PCOS. However, the studies of CYP11A were limited by the failure to make a statistical adjustment for the testing of multiple alleles within the same set of families (Bonferroni correction), an important requirement when performing linkage and association studies of non-Mendelian disorders. In a recent study, the frequency distribution of a polymorphic trinucleotide repeat sequence in exon 1 of the androgen receptor and its pattern of expression via X-inactivation analysis was examined in infertile women with PCOS, fertile controls, and a general population group, all of Australian-Caucasian ethnicity (100). Women with PCOS have a significantly greater frequency of longer CAG alleles and biallelic means (>22 repeats) than the other two subgroups, suggesting that this gene locus may play a role in PCOS pathophysiology. Because PCOS is associated with insulin resistance and glucose intolerance, several investigators have examined the association of PCOS with the insulin gene. A VNTR located in the 5' regulatory region of the insulin gene (INS) on chromosome 11p15.5, which regulates insulin expression and has also been reported to be associated with central obesity (101) and susceptibility to type 2 diabetes (102), was analyzed for a possible role in PCOS (103). Evidence for linkage was examined in 17 families with several affected members. Male pattern baldness was used as the male phenotype (without formal validation). Positive evidence for linkage was obtained (P = 0.002), with the results remaining robust even when male individuals were assigned an "unknown" phenotypic status. In the same report, data from two additional populations also demonstrated that the INS VNTR III/III genotype was associated with an increased risk of PCOS. Interestingly, TDT analysis revealed that class III alleles were transmitted more often from fathers to affected offspring than from mothers. However, association of the insulin gene and PCOS has not been confirmed in larger studies using an even larger numbers of sib pairs (104). Several groups (105, 106, 107) have looked for defects in the insulin receptor gene (INSR), but major mutations have not been discovered. However, 85 Caucasian PCOS patients and 87 age-matched Caucasian control women were recently tested for associations with four candidate genes including follistatin, CYP19 (aromatase), CYP17 (17{alpha} hydroxylase), and the INSR. A microsatellite marker located in the region of the INSR (D19S884) was found to be associated with PCOS (108). Interestingly, of nine additional markers spanning a 7-cM region flanking INSR, no other markers demonstrated a positive association. Although the INSR appears to be a logical candidate gene for PCOS, further testing will be necessary to determine whether there is another gene, closely located to INSR, that is implicated in PCOS. In one of the largest studies to date (104), 37 candidate genes were tested for linkage and association with PCOS or hyperandrogenemia in data from 150 families. Using 39 affected sister pairs, statistically significant evidence for nonparametric linkage was demonstrated in the follistatin gene region, even after appropriate statistical adjustment. However, when DNA sequence variants in the follistatin gene were identified and tested by TDT for association and linkage, no variants demonstrated statistical evidence for linkage when corrected for multiple testing, suggesting that if follistatin does play a role in PCOS, its effect is modest (109). Interestingly, in this data set, the strongest evidence for association was observed in the region of the INSR, but the findings were not statistically significant after correction for multiple tests. Although there is clear evidence that PCOS has a genetic component, based on familial clustering of cases, linkage and association studies have yet to reveal consistent evidence as to the culprit genes for this disorder. Small sample sizes, errors in statistical analysis, and differences in diagnostic criteria between investigators appear to be some of the factors that account for discrepant findings between investigative groups. Inconsistencies aside, molecular genetic approaches still hold promise for elucidating the pathophysiological pathway involved in PCOS; further studies are ongoing.


   VI. Conclusions
Top
 Abstract
 I. Introduction
 II. Commonly Used Genetic...
 III. Use of Unusual...
 IV. Use of Linkage...
 V. Polycystic Ovary Syndrome...
 VI. Conclusions
References
 
The rare, sex-reversed patients leading to the discovery of DAX1, the classic multiplex Finnish families used to map the gene for ODG, and the numerous candidate genes that continue to be tested for association to PCOS are three evolving stories in reproductive medicine. Although perhaps not overtly planned as formal collaborations, each story has been written by a team of clinicians, clinical investigators, and molecular biologists. Determining the sequence of the human genome may further accelerate the pace with which these stories evolve, but the fundamental process of observation and hypothesis formulation remains the same regardless of the tools involved. These are critical lessons to learn—identifying rare and novel phenotypes, delineating the defining features of complex phenotypes, examining enriched populations for inherited disorders, understanding the pros and cons of candidate gene biology, and the challenges inherent in establishing true genotype/phenotype correlations remain the critical frameworks to successful investigation.


   Footnotes
 
Abbreviations: AHC, Adrenal hypoplasia congenita; DAX1, dosage-sensitive, sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome; DSS, dosage-sensitive sex reversal; FSHR, FSH receptor; IHH, idiopathic hypogonadotropic hypogonadism; INS, insulin gene; INSR, insulin receptor; LHR, LH receptor; ODG, ovarian dysgenesis; PCOS, polycystic ovary syndrome; RFLP, restriction fragment length polymorphism; SNP, single nucleotide polymorphism; TDT, transmission-disequilibrium test; VNTR, variable number of tandem repeats.


   References
Top
 Abstract
 I. Introduction
 II. Commonly Used Genetic...
 III. Use of Unusual...
 IV. Use of Linkage...
 V. Polycystic Ovary Syndrome...
 VI. Conclusions
 References
 

  1. Landsteiner K 1900 Zur Kenntnis der antifermentativen, lytischen und agglutinierended Wikungen des Blutserums und der Lymphe. Zbl Bakt 27:357–362
  2. Jeffreys AJ 1975 DNA sequence variants in the G{gamma}-, A{gamma}-, {delta}-, and ß-globin genes of man. Cell 18:1–10
  3. Jeffreys AJ, Wilson V, Thein SL 1985 Hypervariable ‘minisatellite’ regions in human DNA. Nature 314:67–73[CrossRef][Medline]
  4. Weber JL, May PE 1989 Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. Am J Hum Genet 44:388–396[Medline]
  5. Wang DG, Fan JB, Siao CJ, Berno A, Young P, Sapolsky R, Ghandour G, Perkins N, Winchester E, Spencer J, Kruglyak L, Stein L, Hsie L, Topaloglou T, Hubbell E, Robinson E, Mittmann M, Morris MS, Shen N, Kilburn D, Rioux J, Nusbaum C, Rozen S, Hudson TJ, Lipshutz R, Chee M, Lander ES 1998 Large-scale identification, mapping, and genotyping of single-nucleotide polymorphisms in the human genome. Science 280:1077–1082[Abstract/Free Full Text]
  6. Mullikin JC, Hunt SE, Cole CG, Mortimore BJ, Rice CM, Burton J, Matthews LH, Pavitt R, Plumb RW, Sims SK, Ainscough RMR, Attwood J, Bailey JM, Barlow K, Bruskiewich RMM, Butcher PN, Carter NP, Chen Y, Clee CM, Coggill PC, Davies J, Davies RM, Dawson E, Francis MD, Joy AA, Lamble RG, Langford CF, Macarthy J, Mall V, Moreland A, Overton-Larty EK, Ross MT, Smith LC, Steward CA, Ssulston Je, Tinsley EJ, Turney KJ, Willey DL, Wilson GD, McMurray AA, Dunham I, Rogers J, Bentley DR 2000 An SNP map of human chromosome 22. Nature 407:516–519[CrossRef][Medline]
  7. Sachidanandam R, Weissman D, Schmidt SC, Kakol JM, Stein LD, Marth G, Sherry S, Mullikin JC, Mortimore BJ, Willey DL, Hunt SE, Cole CG, Coggill PC, Rice CM, Ning Z, Rogers J, Bentley DR, Kwok P-Y, Mardis ER, Yeh RT, Schultz B, Cook L, Davenport R, Dante M, Fulton L, Hillier L, Waterston RH, McPherson JD, Gilman B, Schaffner S, Van Etten WJ, Reich D, Higgins J, Daly MJ, Blumenstiel B, Baldwin J, Stange-Thomann N, Zody MC, Linton L, Lander ES, Altshuler D 2001 A map of human genome sequence variation containing 1.42 million single nucleotide polymorphisms. Nature 409:928–933[CrossRef][Medline]
  8. Holliday R, Grigg GW 1993 DNA methylation and mutation. Mutat Res 285:61–67[CrossRef][Medline]
  9. Lipshutz R, Fodor SPA, Gingeras TR, Lockhart DJ 1999 High density synthetic oligonucleotide arrays. Nat Genet 21:20–24[CrossRef][Medline]
  10. Suarez BK, Rice J, Reich T 1978 The generalized sib pair IBD distribution: its use in the detection of linkage. Ann Hum Genet 42:87–94[Medline]
  11. Cardon LR, Bell JI 2001 Association study designs for complex diseases. Nature 2:91–99
  12. Greenberg DA 1993 Linkage analysis of "necessary" disease loci versus "susceptibility" loci. Am J Hum Genet 52:135–143[Medline]
  13. Gambaro G, Anglani F, D’Angelo A 2000 Association studies of genetic polymorphisms and complex disease. Lancet 355:308–311[CrossRef][Medline]
  14. Curtis D 1997 Use of siblings in case-control association studies. Ann Hum Genet 61:319–333[CrossRef][Medline]
  15. Spielman RS, McGinnis RE, Ewens WJ 1993 Transmission test for linkage disequilibrium: the insulin gene region and insulin-dependent diabetes mellitus (IDDM). Am J Hum Genet 52:506–516[Medline]
  16. Schulze TG, McMahon FJ 2002 Genetic association mapping at the crossroads: which test and why? Overview and practical guidelines. Am J Med Genet 114:1–11[CrossRef][Medline]
  17. German J, Simpson JL, Chaganti RS, Summitt RL, Reid LB, Merkatz IR 1978 Genetically determined sex-reversal in 46,XY humans. Science 202:53–56[Abstract/Free Full Text]
  18. Narahara K, Kodama Y, Kimura S, Kimoto H 1979 Probable inverted tandem duplication of Xp in a 46,Xp+Y boy. Jap J Hum Genet 24:105–110[CrossRef]
  19. Bernstein R, Jenkins T, Dawson B, Wagner J, Dewald G, Koo GC, Wachtel SS 1980 Female phenotype and multiple abnormalities in sibs with a Y chromosome and partial X chromosome duplication: H–Y antigen and Xg blood group findings. J Med Genet 17:291–300[Abstract/Free Full Text]
  20. Scherer G, Schempp W, Baccichetti C, Lenzini E, Bricarelli FD, Carbone LD, Wolf U 1989 Duplication of an Xp segment that includes the ZFX locus causes sex inversion in man. Hum Genet 81:291–294[CrossRef][Medline]
  21. Ogata T, Hawkins JR, Taylor A, Matsuo N, Hata J, Goodfellow PN 1992 Sex reversal in a child with a 46,X, Yp+ karyotype: support for the existence of a gene(s), located in distal Xp, involved in testis formation. J Med Genet 29:226–230[Abstract/Free Full Text]
  22. Fechner PY, Marcantonio SM, Ogata T, Rosales TO, Smith KD, Goodfellow PN, Migeon CJ, Berkovitz GD 1993 Report of a kindred with X-linked (or autosomal dominant sex-limited) 46,XY partial gonadal dysgenesis. J Clin Endocrinol Metab 76:1248–1253[Abstract]
  23. Bardoni B, Floridia G, Guioli S, Peverali G, Anichini C, Cisternino M, Casalone R, Danesino C, Fraccaro M, Zuffardi O 1993 Functional disomy of Xp22-pter in three males carrying a portion of Xp translocated to Yq. Hum Mol Genet 91:333–338
  24. Bardoni B, Zanaria E, Guioli S, Floridia G, Worley KC, Tonini G, Ferrante E, Chiumello G, McCabe ERB, Fraccaro M, Zuffardi O, Camerino G 1994 A dosage sensitive locus at chromosome Xp21 is involved in male to female sex reversal. Nat Genet 7:497–501[CrossRef][Medline]
  25. Zanaria E, Muscatelli F, Bardoni B, Strom TM, Guioli S, Guo W, Lalli E, Moser C, Walker AP, McCabe ERB, Meitinger T, Monaco AP, Sassone-Corsi P, Camerino G 1994 An unusual member of the nuclear hormone receptor superfamily responsible for X-linked adrenal hypoplasia congenita. Nature 372:635–641[CrossRef][Medline]
  26. Muscatelli F, Strom TM, Walker AP, Zanaria E, Recan D, Meindl A, Bardoni B, Guioli S, Zehetner G, Rabl W, Schwarz HP, Kaplan J, Camerino G, Meitinger T, Monaco AP 1994 Mutations in the DAX-1 gene give rise to both X-linked adrenal hypoplasia congenita and hypogonadotropic hypogonadism. Nature 372:672–676[CrossRef][Medline]
  27. Guo W, Mason JS, Stone Jr CG, Morgan SA, Madu SI, Baldini A, Lindsay EA, Biesecker LG, Copeland KC, Horlick MNB, Pettigrew AL, Zanaria E, McCabe ERB 1995 Diagnosis of X-linked adrenal hypoplasia congenita by mutation analysis of the DAX 1 gene. JAMA 274:324–330[Abstract/Free Full Text]
  28. Nakae J, Tajima T, Kusuda S, Kohda N, Okabe T, Shinohara N, Kato M, Murashita M, Mukai T, Imanaka K, Fujieda K 1996 Truncation at the C-terminus of the DAX-1 protein impairs its biological actions in patients with X-linked adrenal hypoplasia congenita. J Clin Endocrinol Metab 81:3680–3685[Abstract]
  29. Zhang Y-H, Guo W, Wagner RL, Huang B-L, McCabe L, Vilain E, Burris TP, Anyane-Yeboa K, Burghes AHM, Chitayat D, Chudley AE, Genel M, Gertner JM, Klingensmith GJ, Levine SN, Nakamoto J, New MI, Pagon RA, Pappas JG, Quigley CA, Rosenthal IM, Baxter JD, Fletterick RJ, McCabe ERB 1998 DAX1 mutations map to putative structural domains in a deduced three-dimensional model. Am J Hum Genet 62:855–864[CrossRef][Medline]
  30. Reutens AT, Achermann JC, Ito M, Guo W, Habiby RL, Donohoue PA, Pang S, Hindmarsh PC, Jameson JL 1999 Clinical and functional effects of mutations in the DAX-1 gene in patients with adrenal hypoplasia congenita. J Clin Endocrinol Metab 84:504–511[Abstract/Free Full Text]
  31. Swain A, Narvaez V, Burgoyne P, Camerino G, Lovell-Badge R 1998 DAX1 antagonizes Sry action in mammalian sex determination. Nature 391:761–767[CrossRef][Medline]
  32. Guo W, Burris TP, McCabe ER 1995 Expression of DAX-1, the gene responsible for X-linked adrenal hypoplasia congenita and hypogonadotropic hypogonadism, in the hypothalamic-pituitary-adrenal/gonadal axis. Biochem Mol Med 56:8–13[CrossRef][Medline]
  33. Ikeda Y, Swain A, Weber TJ, Hentges KE, Zanaria E, Lalli E, Tamai KT, Sassone-Corsi P, Lovell-Badge R, Camerino G, Parker KL 1996 Steroidogenic factor 1 and Dax-1 colocalize in multiple cell lineages: potential links in endocrine development. Mol Endocrinol 10:1261–1272[Abstract/Free Full Text]
  34. Zazopoulos E, Lalli E, Stocco DM, Sassone-Corsi P 1997 DNA binding and transcriptional repression by DAX-1 blocks steroidogenesis. Nature 390:311–315[CrossRef][Medline]
  35. Lalli E, Bardoni B, Zazopoulos E, Wurtz JM, Strom TM, Moras D, Sassone-Corsi P 1997 A transcriptional silencing domain in DAX-1 whose mutation causes adrenal hypoplasia congenita. Mol Endocrinol 11:1950–1960[Abstract/Free Full Text]
  36. Ito M, Yu R, Jameson JL 1997 DAX-1 inhibits SF-1 mediated transactivation via a carboxy-terminal domain that is deleted in adrenal hypoplasia congenita. Mol Cell Biol 17:1476–1483[Abstract]
  37. Crawford PA, Dorn C, Sadovsky Y, Milbrandt J 1998 Nuclear receptor DAX-1 recruits nuclear receptor corepressor N-CoR to steroidogenic factor 1. Mol Cell Biol 18:2949–2956[Abstract/Free Full Text]
  38. Takahashi T, Shoji Y, Haraguchi N, Takahashi I, Takada G 1997 Active hypothalamic-pituitary-gonadal axis in an infant with X-linked adrenal hypoplasia congenita. J Pediatr 130:485–488[CrossRef][Medline]
  39. Peter M, Matthias V, Partsch C-J, Sippell WG 1998 Congenital adrenal hypoplasia: clinical spectrum, experience with hormonal diagnosis, and report on new point mutations of the DAX-1 gene. J Clin Endocrinol Metab 83:2666–2674[Abstract/Free Full Text]
  40. Seminara SB, Achermann JC, Genel M, Jameson JL, Crowley Jr WF 1999 X-linked adrenal hypoplasia congenita: a mutation in DAX1 expands the phenotypic spectrum in males and females. J Clin Endocrinol Metab 84:4501–4509[Abstract/Free Full Text]
  41. Bassett JH, O’Halloran DJ, Williams GR, Beardwell CG, Shalet SM, Thakker RV 1999 Novel DAX1 mutations in X-linked adrenal hypoplasia congenita and hypogonadotrophic hypogonadism. Clin Endocrinol (Oxf) 50:69–75[CrossRef][Medline]
  42. Kruse K, Sippell WG, Schnakenburg KV 1984 Hypogonadism in congenital adrenal hypoplasia: evidence for a hypothalamic origin. J Clin Endocrinol Metab 58:12–17[Abstract/Free Full Text]
  43. Gordon D, Cohen HN, Beastall GH, Hay ID, Thomson JA 1984 Contrasting effects of subcutaneous pulsatile GnRH therapy in congenital adrenal hypoplasia and Kallmann’s syndrome. Clin Endocrinol (Oxf) 21:597–603[Medline]
  44. Kikuchi K, Kaji M, Momoi T, Mikawa H, Shigematsu Y, Sudo M 1987 Failure to induce puberty in a man with X-linked congenital adrenal hypoplasia and hypogonadotropic hypogonadism by pulsatile administration of low-dose gonadotropin-releasing hormone. Acta Endocrinol (Copenh) 114:153–160[Abstract/Free Full Text]
  45. Bovet P, Reymond MJ, Rey F, Gomez F 1988 Lack of gonadotropic response to pulsatile gonadotropin-releasing hormone in isolated hypogonadotropic hypogonadism associated to congenital adrenal hypoplasia. J Endocrinol Invest 11:201–204[Medline]
  46. Partsch CJ, Sippell WG 1989 Hypothalamic hypogonadism in congenital adrenal hypoplasia. Horm Metab Res 21:623–625[Medline]
  47. Schwartz M, Blichfeldt S, Muller J 1997 X-linked adrenal hypoplasia in a large Greenlandic family. Detection of a missense mutation (N440I) in the DAX-1 gene; implication for genetic counseling and carrier diagnosis. Hum Genet 99:83–87[CrossRef][Medline]
  48. Tabarin A, Achermann JC, Recan D, Bex V, Bertagna X, Christin-Maitre S, Ito M, Jameson JL, Bouchard P 2000 A novel mutation in DAX1 causes delayed-onset adrenal insufficiency and incomplete hypogonadotropic hypogonadism. J Clin Invest 105:321–328[Medline]
  49. Mantovani G, Ozisik G, Achermann JC, Romoli R, Borretta G, Persani L, Spada A, Jameson JL, Beck-Peccoz P 2002 Hypogonadotropic hypogonadism as a presenting feature of late-onset X-linked adrenal hypoplasia congenita. J Clin Endocrinol Metab 87:44–48[Abstract/Free Full Text]
  50. Yu RN, Ito M, Saunders TL, Camper SA, Jameson JL 1998 Role of Ahch in gonadal development and gametogenesis. Nat Genet 20:353–357[CrossRef][Medline]
  51. Jeffs B, Ito M, Yu RN, Martinson FA, Wang ZJ, Doglio LT, Jameson JL 2001 Sertoli cell specific rescue of fertility, but not testicular pathology, in Dax1 (Ahch)-deficient male mice. Endocrinology 142:2481–2488[Abstract/Free Full Text]
  52. Jeffs B, Meeks JJ, Ito M, Martinson FA, Matzuk MM, Jameson JL, Russell LD 2001 Blockage of the rete testis and efferent ductules by ectopic Sertoli and Leydig cells causes infertility in Dax1-deficient male mice. Endocrinology 142:4486–4495[Abstract/Free Full Text]
  53. Merke DP, Tajima T, Baron J, Cutler Jr GB 1999 Hypogonadotropic hypogonadism in a female caused by an X-linked recessive mutation in the DAX1 gene. N Engl J Med 340:1248–1252[Free Full Text]
  54. McCabe ERB 2002 Vulnerability within a robust complex system–DAX-1 mutations and steroidogenic axis development. J Clin Endocrinol Metab 87:41–43[Abstract/Free Full Text]
  55. da Fonte Kohek MB, Batista MC, Russell AJ, Vass K, Giacaglia LR, Mendonca BB, Latronico AC 1998 No evidence of the inactivating mutation (C566T) in the follicle-stimulating hormone receptor gene in Brazilian women with premature ovarian failure. Fertil Steril 70:565–567[CrossRef][Medline]
  56. Whitney EA, Layman LC, Chan PJ, Lee A, Peak DB, McDonough PG 1995 The follicle stimulating hormone receptor gene is polymorphic in premature ovarian failure and normal controls. Fertil Steril 64:518–524[Medline]
  57. Conway GS, Conway E, Walker C, Hoppner W, Gromoll J, Simoni M 1999 Mutation screening and isoform prevalence of the follicle stimulating hormone receptor gene in women with premature ovarian failure, resistant ovary syndrome and polycystic ovary syndrome. Clin Endocrinol (Oxf) 51:97–99[CrossRef][Medline]
  58. Liu JY, Gromoll J, Cedars MI, La Barbera AR 1998 Identification of allelic variants in the follicle-stimulating hormone receptor genes of females with or without hypergonadotropic amenorrhea. Fertil Steril 70:326–331[CrossRef][Medline]
  59. Tuerlings JH, Ligtenberg MJ, Kremer JA, Siers M, Meuleman EJ, Braat DD, Hoefsloot LH, Merkus HM, Brunner HG 1998 Screening male intracytoplasmic sperm injection candidates for mutations of the follicle stimulating hormone receptor gene. Human Reproduction 13:2098–2101[Abstract/Free Full Text]
  60. Simoni M, Gromoll J, Hoppner W, Kamischke A, Krafft T, Stahle D, Nieschlag E 1999 Mutational analysis of the follicle-stimulating hormone (FSH) receptor in normal and infertile men: identification and characterization of two discrete FSH receptor isoforms. J Clin Endocrinol Metab 84:751–755[Abstract/Free Full Text]
  61. Aittomaki K, Lucena JLD, Pakarinen P, Sistonen P, Tapanainen J, Gromoll J, Kaskikari R, Sankila E-M, Lehvaslaiho H, Engel AR, Nieschlag E, Huhtaniemi I, de la Chapelle A 1995 Mutation in the follicle-stimulating hormone receptor gene causes hereditary hypergonadotropic ovarian failure. Cell 82:959–968[CrossRef][Medline]
  62. Simpson JL, Christakos AC, Horwith M, Silverman FS 1971 Gonadal dysgenesis in individuals with apparently normal chromosomal complements: tabulation of cases and compilation of genetic data. Birth Defects Orig Artic Ser 7:215–228[Medline]
  63. Aittomaki K 1994 The genetics of gonadal dysgenesis. Am J Hum Genet 54:844–851[Medline]
  64. Rousseau-Merck MF, Mirashi M, Atger M, Loosfelt H, Milgrom E, Berger R 1990 Localization of the human LH (luteinizing hormone) receptor gene to chromosome 2p21. Cytogenet Cell Genet 54:77–79[Medline]
  65. Rousseau-Merck MF, Atger M, Loosfelt H, Milgrom E, Berger R 1993 The chromosomal localization of the human follicle-stimulating hormone receptor gene (FSHR) on 2p21–2p16 is similar to that of the luteinizing hormone receptor gene. Genomics 15:222–224[CrossRef][Medline]
  66. Kremer H, Mariman E, Otten BJ, Moll GW, Stoelinga GB, Wit JM, Jansen M, Drop SL, Faas B, Ropers HH 1993 Cosegregation of missense mutations of the luteinizing hormone receptor gene with familial male-limited precocious puberty. Hum Mol Genet 2:1779–1783[Abstract/Free Full Text]
  67. Shenker A, Laue L, Kosugi S, Merendino Jr JJ, Minegishi T, Cutler Jr GB 1993 A constitutively activating mutation of the luteinizing hormone receptor in familial male precocious puberty. Nature 365:652–654[CrossRef][Medline]
  68. Yano K, Saji M, Hidaka A, Moriya N, Okuno A, Kohn LD, Cutler Jr GB 1995 A new constitutively activating point mutation in the luteinizing hormone/choriogonadotropin receptor gene in cases of male-limited precocious puberty. J Clin Endocrinol Metab 80:1162–1168[Abstract]
  69. Kremer H, Kraaij R, Toledo SPA, Post M, Fridman JB, Hayashida CY, van Reen M, Milgrom E, Ropers HH, Mariman E, Themmen APN, Brunner HG 1995 Male pseudohermaphroditism due to a homozygous missense mutation of the luteinizing hormone receptor gene. Nat Genet 9:160–164[CrossRef][Medline]
  70. Aittomaki K, Herva R, Stenman UH, Juntunen K, Ylostalo P, Hovatta O, de la Chapelle A 1996 Clinical features of primary ovarian failure caused by a point mutation in the follicle-stimulating hormone receptor gene. J Clin Endocrinol Metab 81:3722–3726[Abstract]
  71. Tapanainen JS, Aittomaki K, Min J, Vaskivuo T, Huhtaniemi IT 1997 Men homozygous for an inactivating mutation of the follicle-stimulating hormone (FSH) receptor gene present variable suppression of spermatogenesis and fertility. Nat Genet 15:205–206[CrossRef][Medline]
  72. Steinberger E 1991 Hormonal control of mammalian spermatogenesis. Physiol Rev 51:1–22
  73. Zirkin BR, Awoniyi C, Griswold MD, Russell LD, Sharpe R 1994 Is FSH required for adult spermatogenesis? J Androl 15:273–276[Free Full Text]
  74. Bremner WJ, Matsumoto AM, Sussman AM, Pauli RM, Paulsen CA 1981 Follicle-stimulating hormone and human spermatogenesis. J Clin Invest 68:1044–1052
  75. O’Driscoll JB, Mamtora H, Higginson J, Pollock A, Kane J, Anderson DC 1994 A prospective study of the prevalence of clear cut endocrine disorders and polycystic ovaries in 350 patients presenting with hirsutism of androgenic alopecia. Clin Endocrinol (Oxf) 41:231–236[Medline]
  76. Polson DW, Adams J, Wadsworth J, Franks S 1988 Polycystic ovaries–a common finding in normal women. Lancet 1:870–872[CrossRef][Medline]
  77. Clayton RN, Ogden V, Hodgkinson J, Worswick L, Rodin DA, Dyer S, Meade TW 1992 How common are polycystic ovaries in normal women and what is their significance for the fertility of the population? Clin Endocrinol (Oxf) 37:127–134[Medline]
  78. Burghen GA, Givens JR, Kitabchi AE 1980 Correlation of hyperandrogenism with hyperinsulinism in polycystic ovarian disease. J Clin Endocrinol 50:113–116[Abstract/Free Full Text]
  79. Dunaif A, Segal KR, Futterweit W, Dobrjansky A 1989 Profound peripheral insulin resistance, independent of obesity, in polycystic ovary syndrome. Diabetes 38:1165–1174[Abstract]
  80. Legro RS, Finegood D, Dunaif A 1998 A fasting glucose to insulin ratio is a useful measure of insulin sensitivity in women with polycystic ovary syndrome. J Clin Endocrinol 83:2694–2698[Abstract/Free Full Text]
  81. Reaven GM 1988 Banting lecture 1988. Role of insulin resistance in human disease. Diabetes 37:1595–1607[Abstract]
  82. Warram JH, Martin BC, Krowleski AS, Soeldner JS, Kahn CR 1990 Slow glucose removal rate and hyperinsulinemia precede the development of type II diabetes in the offspring of diabetic parents. Ann Intern Med 113:909–915
  83. O’Meara NM, Blackman JD, Ehrmann DA, Barnes RB, Jaspan JB, Rosenfield RL, Polonsky KS 1993 Defects in ß-cell function in functional ovarian hyperandrogenism. J Clin Endocrinol Metab 76:1241–1247[Abstract]
  84. Ehrmann DA, Sturis J, Byrne MM, Karrison T, Rosenfield RL, Polonsky KS 1995 Insulin secretory defects in polycystic ovary syndrome. Relationship to insulin sensitivity and family history of non-insulin-dependent diabetes mellitus. J Clin Invest 96:520–527
  85. Dunaif A, Finegood DT 1996 ß-Cell dysfunction independent of obesity and glucose intolerance in the polycystic ovary syndrome. J Clin Endocrinol Metab 81:942–947[Abstract]
  86. Dunaif A 1997 Insulin resistance and the polycystic ovary syndrome: mechanism and implications for pathogenesis. Endocr Rev 18:774–800[Abstract/Free Full Text]
  87. Dahlgren E, Johannson S, Lindstedt G, Knutsson F, Oden A, Janson PO, Mattson LA, Crona N, Lundberg PA 1992 Women with polycystic ovary syndrome wedge resected in 1956 to 1965: a long-term follow-up focusing on natural history and circulating hormones. Fertil Steril 57:505–513[Medline]
  88. Legro RS, Driscoll D, Strauss III JF, Jox J, Dunaif A 1998 Evidence for a genetic basis for hyperandrogenemia in polycystic ovary syndrome. Proc Nat Acad Sci USA 95:14956–14960[Abstract/Free Full Text]
  89. Cooper HE, Spellacy WN, Prem KA, Cohen WD 1968 Hereditary factors in Stein-Leventhal syndrome. Am J Obstet Gynecol 100:371–387[Medline]
  90. Ferriman D, Purdie AW 1979 The inheritance of polycystic ovarian disease and a possible relationship to premature balding. Clin Endocrinol (Oxf) 1:291–299
  91. Lunde O, Magnus P, Sandvik L, Hoglo S 1989 Familial clustering in the polycystic ovarian syndrome. Gynecol Obstet Invest 28:23–30[Medline]
  92. Carey AH, Chan KL, Short F, White D, Williamson R, Franks S 1993 Evidence for a single gene effect causing polycystic ovaries and male pattern baldness. Clin Endocrinol (Oxf) 38:653–659[Medline]
  93. Hague WM, Adams J, Reeders ST, Peto TE, Jacobs HS 1988 Familial polycystic ovaries: a genetic disease? Clin Endocrinol (Oxf) 29:593–605[Medline]
  94. Givens JR 1988 Familial polycystic ovarian disease. Endocrinol Metab Clin North Am 17:771–783[Medline]
  95. Carey AH, Waterworth D, Patel K, White D, Little J, Novelli P, Franks S, Williamson R 1994 Polycystic ovaries and premature male pattern baldness are associated with one allele of the steroid metabolism gene CYP17. Hum Mol Genet 3:1873–1876[Abstract/Free Full Text]
  96. Gharani N, Waterworth D, Williamson R, Franks S 1996 5' Polymorphism of the CYP17 gene is not associated with serum testosterone levels in women with polycystic ovaries. J Clin Endocrinol Metab 81:4174[Free Full Text]
  97. Techatraisak K, Conway GS, Rumsby G 1997 Frequency of a polymorphism in the regulatory region of the 17{alpha}-hydroxylase-17,20-lyase (CYP17) gene in hyperandrogenic states. Clin Endocrinol (Oxf) 46:131–134[CrossRef][Medline]
  98. Franks S 1997 The 17{alpha}-hydroxylase/17,20 lyase gene (CYP17) and polycystic ovary syndrome. Clin Endocrinol (Oxf) 46:135–136[CrossRef][Medline]
  99. Gharani N, Waterworth DM, Batty S, White D, Gilling-Smith C, Conway GS, McCarthy M, Franks S, Williamson R 1997 Association of the steroid synthesis gene CYP11a with polycystic ovary syndrome and hyperandrogenism. Hum Genet 6:397–402[Abstract/Free Full Text]
  100. Hickey T, Chandy A, Norman RJ 2002 The androgen receptor CAG repeat polymorphism and X-chromosome inactivation in Australian Caucasian women with infertility related to polycystic ovary syndrome. J Clin Endocrinol Metab 87:161–165[Abstract/Free Full Text]
  101. Weaver JU, Kopelman PG, Hitman GA 1992 Central obesity and hyperinsulinaemia in women are associated with polymorphism in the 5' flanking region of the human insulin gene. Eur J Clin Invest 22:265–270[Medline]
  102. Bennett ST, Lucassen AM, Gough SCL, Powell EE, Undlein DE, Pritchard LE, Merriman LE, Kawaguchi Y, Dronsfield MJ, Pochiot F, Nerup J, Bouzekri N, Cambon-Thomsen A, Ronningen KS, Barnett AH, Bain SC, Todd JA 1995 Susceptibility to human type 1 diabetes at IDDM2 is determined by tandem repeat variation at the insulin gene minisatellite locus. Nat Genet 9:284–292[CrossRef][Medline]
  103. Waterworth DM, Bennett ST, Gharani N, McCarthy MI, Hague S, Batty S, Conway GS, White D, Todd JA, Franks S, Williamson R 1997 Linkage and association of insulin gene VNTR regulatory polymorphism with polycystic ovary syndrome. Lancet 349:986–990[CrossRef][Medline]
  104. Urbanek M, Legro RS, Driscoll DA, Azziz R, Ehrmann DA, Norman RJ, Strauss III JF, Spielman RS, Dunaif A 1999 Thirty-seven candidate genes for polycystic ovary syndrome: strongest evidence for linkage is with follistatin. Proc Nat Acad Sci USA 96:8573–8578[Abstract/Free Full Text]
  105. Conway GS, Avey C, Rumsby G 1994 The tyrosine kinase domain of the insulin receptor gene is normal in women with hyperinsulinaemia and polycystic ovary syndrome. Human Reproduction 9:1681–1683[Abstract/Free Full Text]
  106. Sorbara LR, Tang Z, Cama A, Xia J, Schenker E, Kohanski RA, Poretsky L, Koller E, Taylor SI, Dunaif A 1994 Absence of insulin receptor gene mutations in three insulin-resistant women with the polycystic ovary syndrome. Metabolism 43:1568–1574[CrossRef][Medline]
  107. Talbot JA, Bicknell EJ, Rajkhowa M, Krook A, O’Rahilly S, Clayton RN 1996 Molecular scanning of the insulin receptor gene in women with polycystic ovarian syndrome. J Clin Endocrinol Metab 81:1979–1983[Abstract]
  108. Tucci S, Futterweit W, Concepcion ES, Greenberg DA, Villanueva R, Davies TF, Tomer Y 2001 Evidence for association of polycystic ovary syndrome in Caucasian women with a marker at the insulin receptor gene locus. J Clin Endocrinol Metab 86:446–449[Abstract/Free Full Text]
  109. Urbanek M, Wu X, Vickery KR, Kao L-C, Christenson LK, Schneyer A, Legro RS, Driscoll DA, Strauss III JF, Dunaif A, Spielman RS 2000 Allelic variants of the follistatin gene in polycystic ovary syndrome. J Clin Endocrinol Metab 85:4455–4461[Abstract/Free Full Text]



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