Genetic markers of repolarization and arrhythmic events after acute coronary syndromes.

BACKGROUND: There is a genetic contribution to the risk of ventricular arrhythmias in survivors of acute coronary syndromes (ACS). We wished to explore the role of 33 candidate single nucleotide polymorphisms (SNPs) in prolonged repolarization and sudden death in patients surviving ACS. METHODS: A total of 2,139 patients (1680 white ethnicity) surviving an admission for ACS were enrolled in the prospective Coronary Disease Cohort Study. Extensive clinical, echocardiographic, and neurohormonal data were collected for 12 months, and clinical events were recorded for a median of 5 years. Each SNP was assessed for association with sudden cardiac death (SCD)/cardiac arrest (CA) and prolonged repolarization at 3 time-points: index admission, 1 month, and 12 months postdischarge. RESULTS: One hundred six SCD/CA events occurred during follow-up (6.3%). Three SNPs from 3 genes (rs17779747 [KCNJ2], rs876188 [C14orf64], rs3864180 [GPC5]) were significantly associated with SCD/CA in multivariable models (after correction for multiple testing); the minor allele of rs17779747 with a decreased risk (hazard ratio [HR] 0.68 per copy of the minor allele, 95% CI 0.50-0.92, P = .012), and rs876188 and rs386418 with an increased risk (HR 1.52 [95% CI 1.10-2.09, P = .011] and HR 1.34 [95% CI 1.04-1.82, P = .023], respectively). At 12 months postdischarge, rs10494366 and rs12143842 (NOS1AP) were significant predictors of prolonged repolarization (HR 1.32 [95% CI 1.04-1.67, P = .022] and HR 1.30 [95% CI 1.01-1.66, P = .038], respectively), but not at earlier time-points. CONCLUSION: Three SNPs were associated with SCD/CA. Repolarization time was associated with variation in the NOS1AP gene. This study demonstrates a possible role for SNPs in risk stratification for arrhythmic events after ACS.

FOXL2 copy number changes in the molecular pathogenesis of BPES: unique cohort of 17 deletions.

Blepharophimosis Syndrome (BPES) is an autosomal dominant developmental disorder of the eyelids with or without ovarian dysfunction caused by FOXL2 mutations. Overall, FOXL2deletions represent 12% of all genetic defects in BPES. Here, we have identified and characterized 16 new and one known FOXL2 deletion combining multiplex ligation-dependent probe amplification (MLPA), custom-made quantitative PCR (qPCR) and/or microarray-based copy number screening. The deletion breakpoints could be localized for 13 out of 17 deletions. The deletion size is highly variable (29.8 kb - 11.5 Mb), indicating absence of a recombination hotspot. Although the heterogeneity of their size and breakpoints is not reflected in the uniform BPES phenotype, there is considerable phenotypic variability regarding associated clinical findings including psychomotor retardation (8/17), microcephaly (6/17), and subtle skeletal features (2/17). In addition, in all females in whom ovarian function could be assessed, FOXL2 deletions proved to be associated with variable degrees of ovarian dysfunction. In conclusion, we present the largest series of BPES patients with FOXL2 deletions and standardized phenotyping reported so far. Our genotype-phenotype data can be useful for providing a prognosis (i.e. occurrence of associated features) in newborns with BPES carrying a FOXL2 deletion.

Zebrafish as a model for long QT syndrome: the evidence and the means of manipulating zebrafish gene expression.

Congenital long QT syndrome (LQT) is a group of cardiac disorders associated with the dysfunction of cardiac ion channels. It is characterized by prolongation of the QT-interval, episodes of syncope and even sudden death. Individuals may remain asymptomatic for most of their lives while others present with severe symptoms. This heterogeneity in phenotype makes diagnosis difficult with a greater emphasis on more targeted therapy. As a means of understanding the molecular mechanisms underlying LQT syndrome, evaluating the effect of modifier genes on disease severity as well as to test new therapies, the development of model systems remains an important research tool. Mice have predominantly been the animal model of choice for cardiac arrhythmia research, but there have been varying degrees of success in recapitulating the human symptoms; the mouse cardiac action potential (AP) and surface electrocardiograms exhibit major differences from those of the human heart. Against this background, the zebrafish is an emerging vertebrate disease modelling species that offers advantages in analysing LQT syndrome, not least because its cardiac AP much more closely resembles that of the human. This article highlights the use and potential of this species in LQT syndrome modelling, and as a platform for the in vivo assessment of putative disease-causing mutations in LQT genes, and of therapeutic interventions.

Posthumous diagnosis of long QT syndrome from neonatal screening cards.

BACKGROUND: Molecular autopsy in sudden unexplained death in the young (SUDY) victims cannot usually be performed if tissue suitable for DNA extraction is not retained at autopsy. OBJECTIVE: The purpose of this study was to assess the feasibility and clinical value of posthumous genetic testing for long QT syndrome (LQTS) using residual material from the neonatal screening (Guthrie) card in SUDY victims. METHODS: Twenty-one cases were investigated up to 13 years after death. Deaths occurred at <1 year in one, 1-18 years in 18, and 19-35 years in two patients. Guthrie cards were 3-39 years old. DNA was extracted, and amplicons corresponding to the coding regions of the LQTS genes 1, 2, 3, 5, and 6 underwent either denaturing high-performance liquid chromatography screening or direct DNA sequencing. RESULTS: Adequate DNA was extracted in every case, although repeated purification and amplification was often required. Rare variants were detected in six of 19 cases undergoing diagnostic screening. Four (21%) are considered to be pathological and have been used for family screening: R243C and H455Y in KCNQ1 in 12-year-old and 13-year-old boys, respectively, and Q81H and S621R in KCNH2 in 21-month and 28-year-old females, respectively. Variants of uncertain significance were R1047L in KCNH2 in a 2-year-old girl and S38G in KCNE1 in a 19-month-old boy. Point mutation tests for previously identified familial LQTS mutations revealed a positive result in both cases: E146K in KCNQ1 and exon 6-4del in KCNH2. CONCLUSION: Residual material from Guthrie cards collected for newborn metabolic screening can be used as a reliable source of DNA for the posthumous diagnosis of LQTS decades after SUDY, although purification and amplification of DNA is time intensive.

Inhibin and premature ovarian failure.

BACKGROUND: Elucidation of the causes of premature ovarian failure (POF) is difficult due to the heterogeneity of the condition. Inhibin is a potential candidate gene for POF based on its dual actions on FSH secretion by the pituitary and gametogenesis in the gonads. A missense mutation in the inhibin alpha subunit gene (INHA G769A) is associated with POF in several populations. However, there is phenotypic heterogeneity in INHA G769A mutation carriers. METHODS: Relevant studies were identified by searching PubMed and mutational frequencies combined for meta-analysis. RESULTS: Meta-analysis of published studies revealed a risk difference of 0.04 (-0.030 to 0.11). The occurrence of asymptomatic carriers in populations suggests incomplete penetrance and/or a multi-genetic cause of POF. We propose that a decline in inhibin bioactivity caused by the mutation could increase FSH levels; and in a susceptible individual, the heightened sensitivity to gonadotrophins causes POF. Impaired paracrine effects of inhibin could impact folliculogenesis due to reduced antagonism of activin, bone morphogenetic protein 15 and growth differentiation factor 9. Functional studies of this mutation indicate normal production of dimeric inhibin A and B and impaired bioactivity of inhibin B. CONCLUSIONS: The identification of an autosomal mutation in the inhibin alpha subunit gene that is significantly linked to POF in certain ethnic populations highlights the role of inhibin in the regulation of ovarian biology and fertility. Although the reduction of inhibin B bioactivity by the INHA G769A mutation is clearly not the only cause, evidence suggests that this change may serve as a susceptibility factor, increasing the likelihood of POF.

Single nucleotide polymorphisms in human Paneth cell defensin A5 may confer susceptibility to inflammatory bowel disease in a New Zealand Caucasian population.

BACKGROUND: Human Paneth cell alpha-defensins, especially DEFA5, are involved in maintaining homeostasis of the human microbial microflora. Since breakdown of normal mucosal antibacterial defence occurs in inflammatory bowel disease (IBD), variants in the DEFA5 gene could be associated with IBD risk. SUBJECTS: A cohort of 25 patients with indeterminate colitis (IC), 405 with ulcerative colitis (UC), and 385 with Crohn's disease (CD), were compared with 201 control individuals from the Canterbury region in New Zealand. METHODS: A 15 kb haplotype block surrounding DEFA5 contained 35 HapMap markers which were polymorphic in Caucasians. Four markers (A-D) were selected to tag 27 of the 35 markers at r(2)>0.68, and were genotyped in DNA samples. RESULTS: Minor allele frequencies for all single nucleotide polymorphisms (SNPs) were somewhat elevated in patients. Subgroup analysis showed SNP A had odds ratio 1.44 in UC patients with pancolitis (95% C.I. 1.07-1.94), SNP B odds ratio 2.37 in CD patients with onset prior to 17 years age (95% C.I. 1.12-5.03), SNP C odds ratio 1.68 in UC patients with left colonic localisation (95% C.I. 1.12-2.52), and SNP D had odds ratio 1.56 in CD patients with one or more relatives with IBD (95% C.I. 1.03-2.35). Two two-marker haplotypes and one three-marker haplotype were associated with UC (p-values 0.025-0.05). CONCLUSIONS: The SNPs genotyped in our study were surrogates for common variants, and observed associations between these and IBD status are likely due to linkage disequilibrium with a functional common DEFA5 variant. Identifying such functional variants will be prioritized in subsequent work.

Sex of bovine embryos may be related to mothers' preovulatory follicular testosterone.

Although the sex of the offspring in mammals is commonly viewed as a matter of chance (depending on whether an X or a Y chromosome-bearing spermatozoon reaches the ovum first), evolutionary biologists have shown that offspring sex ratios are often significantly related to maternal dominance, a characteristic that has been shown to be linked to testosterone in female mammals, including humans. Hence, we hypothesized that variations in female testosterone might be related to reproductive mechanisms associated with sex determination, with higher levels of follicular testosterone being associated with a greater likelihood of conceiving a male. To investigate this hypothesis we collected follicular fluid and cumulus-oocyte complexes from bovine antral follicles. Individual matched samples of follicular fluid were assayed for testosterone, whereas the oocytes were matured, fertilized, and cultured in vitro. The resultant embryos were sexed by PCR. The level of testosterone in the follicular fluid was then compared with sex of the embryo (n = 171). Results showed that follicular testosterone levels were significantly higher for subsequently male embryos (Mann-Whitney U = 2823; P [one-tailed] = 0.016). When we excluded embryos from follicles in which the estradiol-to-testosterone ratio was more than 1 (leaving a sample size of 135), the same result held (Mann-Whitney U = 1667; P [one-tailed] = 0.009). Thus, bovine ova that developed in follicular fluid with high concentrations of testosterone in vivo were significantly more likely to be fertilized by Y chromosome-bearing spermatozoa.

Gender-stratified analysis of DLG5 R30Q in 4707 patients with Crohn disease and 4973 controls from 12 Caucasian cohorts.

BACKGROUND: DLG5 p.R30Q has been reported to be associated with Crohn disease (CD), but this association has not been replicated in most studies. A recent analysis of gender-stratified data from two case-control studies and two population cohorts found an association of DLG5 30Q with increased risk of CD in men but not in women and found differences between 30Q population frequencies for males and females. Male-female differences in population allele frequencies and male-specific risk could explain the difficulty in replicating the association with CD. METHODS: DLG5 R30Q genotype data were collected for patients with CD and controls from 11 studies that did not include gender-stratified allele counts in their published reports and tested for male-female frequency differences in controls and for case-control frequency differences in men and in women. RESULTS: The data showed no male-female allele frequency differences in controls. An exact conditional test gave marginal evidence that 30Q is associated with decreased risk of CD in women (p = 0.049, OR = 0.87, 95% CI 0.77 to 1.00). There was also a trend towards reduced 30Q frequencies in male patients with CD compared with male controls, but this was not significant at the 0.05 level (p = 0.058, OR = 0.87, 95% CI 0.74 to 1.01). When data from this study were combined with previously published, gender-stratified data, the 30Q allele was found to be associated with decreased risk of CD in women (p = 0.010, OR = 0.86, 95% CI 0.76 to 0.97), but not in men. CONCLUSION: DLG5 30Q is associated with a small reduction in risk of CD in women.

Ovarian reserve tests for predicting fertility outcomes for assisted reproductive technology: the International Systematic Collaboration of Ovarian Reserve Evaluation protocol for a systematic review of ovarian reserve test accuracy.

BACKGROUND: The presence of a wide range of tests of ovarian reserve suggests that no single test provides a sufficiently accurate result. Many tests are used without reference to an evidence base. So far, individual studies conducted on these tests are too small to give precise estimates of prognostic accuracy. OBJECTIVES: To systematically assess the accuracy of the available tests of ovarian reserve in terms of prediction of fertility outcomes. SEARCH STRATEGY: The search will be conducted using the name of the respective index test being studied (as listed on the MESH database), if more than 2000 citations are listed, 'ovary' and or 'ovarian', 'fertility' and or 'reserve' will be combined with the original search term as required. Studies of the accuracy of tests of ovarian reserve will be obtained without language restrictions from 1980 to 2005 using the following electronic databases and Ovid software: MEDLINE, EMBASE, PUBmed, Biological extracts, Pascal, Cochrane Library (CDSR, DARE, CCTR, HTA), Best Evidence databases, SCISEARCH, Conference Proceedings (ISI Proceedings, Healthstar, Current Contents, Science Citation Index, Cancerlit and Econlit and NHS Economic Evaluation database. The National Research Register, the Medical Research Council's Clinical Trials Register, MEDION, DARE, and the US Clinical Trials register. SELECTION CRITERIA: Studies will be selected if accuracy of tests are compared with a reference standard and include data that can be abstracted into a two-by-two table to calculate sensitivity and specificity. The studies to be included in this review will examine one of the following index 'tests' within a study population of women undergoing assisted reproductive technology: * Clinical variables--age, history of cancelled cycles. * Basal blood tests--follicle-stimulating hormone (FSH), lutenising hormone (LH), FSH:LH ratios, estradiol (E(2)), inhibin A and B, progesterone (P(4)), P(4):E(2) ratios, antimullerian hormone, testosterone, vascular endothelial growth factor, insulin-like growth factor-1:insulin-like growth factor binding protein-1 ratios. * Dynamic tests--clomiphene citrate challenge test, gonadotropin analogue stimulating test, exogenous FSH ovarian reserve test. * Ultrasound tests-antral follicle count, ovarian volume, ovarian stromal peak systolic velocity, including waveform and pulsatility index, ovarian follicular vascularity. * Histology--ovarian biopsy. Data collection and analysis Two independent reviewers will perform quality assessment and data extraction. Prognostic accuracy will be determined by calculating positive and negative likelihood ratios for the following outcomes or reference standards: live birth, ongoing pregnancy, clinical pregnancy, biochemical pregnancy, embryos available for transfer, eggs obtained at oocyte retrieval, cycles cancelled prior to oocyte retrieval. Main results and conclusions N/A.

Expression of TGF-beta1, TGF-beta2, TGF-beta3 and the receptors TGF-betaRI and TGF-betaRII in placentomes of artificially inseminated and nuclear transfer derived bovine pregnancies.

Bovine nuclear transfer pregnancies are characterized by a high incidence of placental abnormalities, notably, increased placentome size and deficiencies in trophoblast cell function and establishment of placental vasculature. Alterations in gene expression during placental growth and development may contribute to the appearance of large placentomes in pregnancies derived from nuclear transfer. The placenta synthesizes a number of cytokines and growth factors, including the transforming growth factor-betas (TGF-betas) that are involved in the establishment, maintenance and/or regulation of pregnancy. All forms of TGF-beta and their receptors are present at the fetal-maternal interface of the bovine placentome, where they are thought to play an important role in regulating growth, differentiation, and function of the placenta. Using real-time RT-PCR, we have examined the expression of TGF-beta1, TGF-beta2, TGF-beta3 and the receptors TGF-betaRI and TGF-betaRII in placentomes of artificially inseminated (AI) and nuclear transfer (NT)-derived bovine pregnancies at days 50, 100 and 150 of gestation. TGF-beta1, TGF-beta2 and TGF-beta3 mRNA expression increased by 2.0-2.8-fold, while TGF-betaRI and TGF-betaRII mRNA expression decreased by 1.7-2.0-fold in NT placentomes compared to AI controls at all gestational ages examined. These findings indicate that NT placentomes may be resistant to the growth suppressive effects of TGF-betas and could contribute to the placental proliferative abnormalities observed in NT-derived placentas. Alternatively, deficiencies in placentation may provide a mechanism whereby TGF-betas are dysregulated in NT pregnancies.

Blepharophimosis and bilateral Duane syndrome associated with a FOXL2 mutation.

This case describes the novel coexistence of sporadic blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) and bilateral type I Duane syndrome in a female infant, with a FOXL2 mutation. Mutational analysis of FOXL2 demonstrated a 30-nucleotide duplication (c.672(-)701dup30) within the polyalanine tract of FOXL2. The association of BPES and Duane syndrome represents a novel phenotype which may suggest a greater pleiotropic effect of FOXL2 in development. During the period of the 4-8th week of embryonic development, the cranial nerves, their nuclei and the corresponding innervation to the extraocular muscles develop, the extraocular muscles undergo development and differentiation. This coincides with the period of time that FOXL2 is expressed strongly in the developing eyelids and the surrounding tissues. Forkhead genes are transcription factors and likely to be involved in signal transduction pathways. This case expands the spectrum of FOXL2 mutations associated with BPES.

Epigenetic regulation of inhibin alpha-subunit gene in prostate cancer cell lines.

Inhibin was first identified as a gonad-derived regulator of pituitary FSH; however, it has subsequently been shown to be a tumour suppressor in the gonad and adrenal glands. Whereas non-malignant regions of human primary prostate carcinomas express inhibin alpha-subunit (INHA), malignant tissues lack INHA transcript and protein, which is consistent with epigenetic regulation of the inhibin alpha-subunit gene (INHA) promoter. This study investigated whether methylation of the INHA promoter was responsible for inactivation of INHA transcription and translation in the prostate cancer cell lines, LNCaP, DU145 and PC3. Methylation of the promoter was revealed by bisulphite genomic sequencing and use of inhibitors of methylation and histone deacetylation resulted in reactivation of the INHA transcription and translation. Significant (P<0.05) downregulation of a luciferase reporter gene downstream from a methylated INHA promoter compared with unmethylated INHA promoter occurred in vitro. The data demonstrate that promoter methylation is associated with downregulation of the INHA gene in prostate cancer cell lines, which is consistent with its tumour suppressive role. Therefore INHA has a significant role in prostate tumorigenesis.

SPRASA, a novel sperm protein involved in immune-mediated infertility.

BACKGROUND: Antisperm antibodies (ASA) may be an important cause of infertility, but current tests for the detection of ASA have poor prognostic value. Identification of the sperm proteins that ASA bind to may aid the development of more useful diagnostic tests. METHODS: One- and two-dimensional PAGE and western blotting analyses, as well as amino acid sequencing, were used to identify a novel sperm protein reactive with ASA (SPRASA) from infertile men. An antiserum reactive with SPRASA was produced by immunizing a rabbit with SPRASA excised from two-dimensional gels. This antiserum was used to demonstrate the localization of SPRASA on the sperm. RESULTS: Amino acid sequences derived from SPRASA matched those of a theoretical protein, XP-085564. This protein is derived from the C-type lysozyme/alpha-lactalbumin gene family. Immunohisto chemistry indicates that SPRASA is localized to the acrosome. Western blot analysis revealed that 50 unselected individuals did not have antibodies that reacted with SPRASA. CONCLUSION: Only ASA from infertile men react with SPRASA, suggesting that this novel protein may be important in the processes of fertility. The identification of SPRASA as the antigen for infertility-associated ASA raises the possibility of developing first, antigen-specific tests for ASA, and secondly, more targeted treatment for immune-mediated infertility.

Activity, synthesis, storage, and messenger RNA of cyclooxygenase in intrauterine tissues of guinea pigs near term and during labor.

Whether the reported gestation-dependent increase in cyclooxygenase activity in gestational tissues is due to an accumulation of cyclooxygenase in vivo or an increasing capacity to synthesize cyclooxygenase in vitro is unknown. In this study in guinea pigs, COX activity was estimated from the net production rates of prostaglandins E(2) and F(2alpha) in the presence of optimal substrate concentrations. Cyclooxygenase activity in amnion increased between 45 days of gestation and labor in microsomes (150-fold in relation to PGF(2alpha) production and 116-fold in relation to PGE(2) production) and in tissue explants (42-fold in relation to PGF(2alpha) production). The capacity for de novo synthesis of cyclooxygenase after aspirin treatment increased nine-fold between 45 days of gestation and labor in amnion explants. Comparison of COX activity in amnion explants with or without prior aspirin treatment showed that COX activity is at least three-fold higher in controls than would be expected if the activity was due to de novo synthesis alone. Cyclooxygenase-2 mRNA predominated in amnion but neither cyclooxygenase-2 nor cyclooxygenase-1 mRNA levels (semi-quantitative RT-PCR) changed significantly. This suggests that the gestation-dependent increase in cyclooxygenase activity in guinea pig amnion is due in part to accumulation of cyclooxygenase in vivo, that COX-2 predominates, and that COX activity is not correlated with levels of COX mRNA.

Identification of novel mutations in FOXL2 associated with premature ovarian failure.

Premature ovarian failure (POF) affects approximately 1% of women and is known to be caused by sex chromosome abnormalities, iatrogenic agents and autoimmune diseases, but in the majority of cases the cause is unknown. However, several families have been identified as having an inherited predisposition to POF, suggesting a genetic component to the condition in these cases. The FOXL2 gene of 70 POF patients from New Zealand and Slovenia was screened for mutations. In a Slovenian POF patient, a novel 30 bp deletion was identified that was predicted to remove 10 out of 14 alanines (A221_A230del), from the polyalanine tract downstream of the winged helix/forkhead domain of the FOXL2 protein. A novel single nucleotide substitution, 772(1009)T>A, which is predicted to change amino acid 258 from tyrosine to asparagine (Y258N), was identified in a New Zealand POF patient. Neither mutation was identified in 200 normal control chromosomes from 100 control samples. Three previously unreported single nucleotide substitutions, considered to be non-functional polymorphisms, were also identified.

Molecular genetics of ovarian cancer.

This is a review of the approaches that can be used to analyze genetic changes in ovarian cancer. Traditional gene localization methods are discussed, followed by a section on gene identification techniques. Once a putative disease-associated gene has been cloned, mutations have to be identified and analyzed. There are numerous mutation detection methods, and the most common ones are outlined. In the penultimate section, the role of immunohistochemistry as a surrogate method for mutation analysis is considered. Finally, the possible use of functional assays is discussed. At present, it would appear that DNA chip technology for the detection of mutations, and microarray analysis of gene expression, are two important techniques likely to have a significant impact on the genetic analysis of ovarian cancer.

Analysis of the TGF beta functional pathway in epithelial ovarian carcinoma.

Epithelial ovarian carcinoma is often diagnosed at an advanced stage of disease and is the leading cause of death from gynaecological neoplasia. The genetic changes that occur during the development of this carcinoma are poorly understood. It has been proposed that IGFIIR, TGFbeta1 and TGFbetaRII act as a functional unit in the TGFbeta growth inhibitory pathway, and that somatic loss-of-function mutations in any one of these genes could lead to disruption of the pathway and subsequent loss of cell cycle control. We have examined these 3 genes in 25 epithelial ovarian carcinomas using single-stranded conformational polymorphism analysis and DNA sequence analysis. A total of 3 somatic missense mutations were found in the TGFbetaRII gene, but none in IGFRII or TGFbeta1. An association was found between TGFbetaRII mutations and histology, with 2 out of 3 clear cell carcinomas having TGFbetaRII mutations. This data supports other evidence from mutational analysis of the PTEN and beta-catenin genes that there are distinct developmental pathways responsible for the progression of different epithelial ovarian cancer histologic subtypes.

Molecular genetics of ovarian cancer : a technical overview.

This chapter is an overview, from a technical perspective, of the approaches that can be used to analyze genetic changes in ovarian cancers. Traditional gene localization methods are discussed, followed by a section on gene identification techniques. Once a putative disease-associated gene has been cloned, mutations have to be identified and analyzed. There are numerous mutation detection methods, and the most common ones are outlined. In the penultimate section, the role of immunohistochemistry as a surrogate method for mutation analysis is considered. Finally, the possible use of functional assays is discussed. The number of techniques used in the molecular analysis of ovarian cancer is immense, and it is beyond the scope of this book to describe all of these methods. However, the most important methods have been outlined in this overview chapter, and many are described in detail in the following chapters.

Inhibin: a candidate gene for premature ovarian failure.

Premature ovarian failure (POF) occurs in 1% of all women, and in 0.1% of women under the age of 30 years. The mechanisms that give rise to POF are largely unknown. Inhibin has a role in regulating the pituitary secretion of FSH, and is therefore a potential candidate gene for ovarian failure. Using single-stranded conformation polymorphism (SSCP) and DNA sequencing, DNA samples were screened from 43 women with POF for mutations in the three inhibin genes. Two variants were found: a 1032C-->T transition in the INHssA gene in one patient, and a 769G-->A transition in the INHalpha gene in three patients. The INHssA variant appears to be a polymorphism, as there was no change in the amino acid sequence of the gene product. The INHalpha variant resulted in a non-conservative amino acid change, with a substitution from alanine to threonine. This alanine is highly conserved across species, and has the potential to affect receptor binding. The INHalpha variant is significantly associated with POF (3/43 patients; 7%) compared with control samples (1/150 normal controls; 0.7%) (Fisher's exact test, P < 0.035). Further analysis of the inhibin gene in POF patients and matched controls will determine its role in the aetiology of POF.