Mutation analysis of two candidate genes for premature ovarian failure, DACH2 and POF1B

BACKGROUND: Balanced X;autosome translocations interrupting the 'critical region' of the long arm of the human X chromosome are often associated with premature ovarian failure (POF). However, the mechanisms leading to X-linked ovarian dysfunction are largely unknown, as the majority of the X chromosome breakpoints have been mapped to gene-free genomic regions. A few genes have been found to be interrupted, but their role has never been clarified. METHODS AND RESULTS: By fine mapping of the X chromosome breakpoint of an X;autosome balanced translocation, we identified a new interrupted gene, POF1B. We performed a mutation analysis of POF1B and of another gene previously identified, DACH2, localized approximately 700 kb distal in Xq21, in a cohort of >200 Italian POF patients. Rare mutations were found in patients in both genes. CONCLUSIONS: Our findings could not demonstrate any involvement of POF1B, but suggest that rare mutations in the DACH2 gene may have a role in the POF phenotype.     

A susceptibility locus rs7099208 is associated with non-obstructive azoospermia via reduction in the expression of FAM160B1

Non-obstructive azoospermia (NOA) is a severe defect in male reproductive health that occurs in 1% of adult men. In a previous study, we identified that rs7099208 is located within the last intron of FAM160B1 at 10q25.3. In this study, we analysed expression Quantitative Trait Loci (eQTL) of FAM160B1, ABLIM1 and TRUB1, the three genes surrounding rs7099208. Only the expression level of FAM160B1 was reduced for the homozygous alternate genotype (GG) of rs7099208, but not for the homozygous reference or heterozygous genotypes. FAM160B1 is predominantly expressed in human testes, where it is found in spermatocytes and round spermatids. From 17 patients with NOA and five with obstructive azoospermia (OA), immunohistochemistry revealed that expression of FAM160B1 is reduced, or undetectable in NOA patients, but not in OA cases or normal men. We conclude that rs7099208 is associated with NOA via a reduction in the expression of FAM160B1.     

The association of FMR1 gene (CGG)n variation with idiopathic female infertility

IntroductionThe FMR1 gene plays an important role in brain development and in the regulation of ovarian function. The FMR1 gene contains CGG repeat variation and the expansion of the repeats is associated with various phenotypes e.g. fragile X syndrome, premature ovarian failure, etc. Repeats ranging < 55 CGG are considered normal, however recent studies suggest that high-normal (35–54 CGG) and low-normal (< 26 CGG) alleles may also have an impact on female reproductive function.Material and methodsWe have performed a case-control study to assess the impact of FMR1 gene CGG repeats on female infertility. The study comprised 161 women with primary and secondary idiopathic infertility and 12 females with diminished ovarian reserve. The control group consisted of 129 healthy women with children. The FMR1 gene trinucleotide CGG repeat variation was detected using a triplet repeat primed polymerase chain reaction with capillary electrophoresis.ResultsThe analysis of CGG repeats revealed that high-normal alleles are statistically significantly more common in the secondary infertility group than in controls (12% vs. 4.3%, p = 0.03, OR = 3.1, 95% CI: 1.1–8.3). The distribution of high-normal alleles and genotypes did not differ between patients with primary infertility and controls (p > 0.05). In addition, the analysis of low-normal allele and genotype frequencies did not present a difference between primary, secondary infertility and the control group (p > 0.05).ConclusionsIn our study, the FMR1 gene high-normal alleles were associated with secondary infertility. However, to address the controversies related to the role of FMR1 genes in the development of diminished ovarian reserve, further studies on the subject are required.     

A novel mutation in FK506 binding protein-like (FKBPL) causes male infertility

AimTo perform a mutation analysis of FK506 binding protein-like (FKBPL) in patients with azoospermia.MethodsDNA samples were isolated from the peripheral blood of 30 azoospermic male patients with normal 46 XY karyotype and 10 healthy controls. Multiplex polymerase chain reaction assays were used to evaluate Y microdeletions, and the patients without deletions were further analyzed. Sanger sequencing was used for mutation analysis.ResultsA heterozygous adenine to guanine substitution was observed at position c.28 (c.28A>G) (one patient), guanine to adenine substitution at c.90 (c.90G>A) (three patients), and a novel insertion mutation of TCTCATAAGTCT at c. 229_240dup (two patients), all in FKBPL exon 2. Furthermore, four different benign variants were observed in the same gene.ConclusionOur study supports the literature on the etiologic effects of changes on autosomal chromosomes and highlights the importance of molecular analysis of all known and unknown genes that could be involved in male sexual development and function.

Genetic alterations are implicated in the pathogenesis of male infertility in 10%-15% of patients (1). The most common cause of azoospermia are microdeletions of the AZF region on the Y chromosome (2,3). Only a small proportion of other pathogenic variants in azoospermia cases have been identified, and other genetic causes need to be explored (4). In most infertility clinics, chromosome Y microdeletion analysis is the last step in molecular male infertility diagnostics (5). However, this analysis alone is insufficient since male infertility is related to complex genetic alterations.To date, many genes have been investigated for their effect on spermatogenesis. As a result, many pathogenic and normal variants that affect protein expression, structure, and function in spermatogenesis have been identified and associated with azoospermia infertility. However, the studies are still insufficient in terms of determining the diagnostic factors, and infertility remains a major problem worldwide. Genetic alterations of autosomal genes could be emerging causes of infertility in azoospermic men negative for Y microdeletions.Linkage analysis and clustering of chromosomal breakpoints in infertile men showed that FK506-binding protein like (FKBPL) gene pathogenic variants play an important role in azoospermia (6,7). FKBPL, a member of the FKBP family (DIR1, WISp39), was first described in 1999 and is chromosomally localized at the position 6p21.3. It is strongly expressed in the testis, including the Sertoli and Leydig cells (8,9). Pathogenic variants in FKBPL cause male infertility by disrupting the androgen receptor (AR) signal. FKBPL has been reported to increase the transactivation of AR-responsive genes.The aim of this study was to investigate whether the pathogenic variant analysis of FKBPL could be used as a routine test in azoospermic men negative for Y microdeletions, since autosomal genes are not routinely tested.     

ESR1 rs9340799 Is Associated with Endometriosis-Related Infertility and In Vitro Fertilization Failure

Estrogen receptor alpha has a central role in human fertility by regulating estrogen action in all human reproductive tissues. Leukemia inhibitory factor (LIF) expression, a cytokine critical for blastocyst implantation, is mediated by estrogen signaling, so we hypothesized that ESR1 gene polymorphisms might be candidate risk markers for endometriosis-related infertility and in vitro fertilization (IVF) failure. We included 98 infertile women with endometriosis, 115 infertile women with at least one IVF failure and also 134 fertile women as controls. TaqMan SNP assays were used for genotyping LIF (rs929271), MDM2 (rs2279744), MDM4 (rs1563828), USP7 (rs1529916), and ESR1 (rs9340799 and rs2234693) polymorphisms. The SNP ESR1 rs9340799 was associated with endometriosis-related infertility (P < 0.001) and also with IVF failure (P = 0.018). After controlling for age, infertile women with ESR1 rs9340799 GG genotype presented 4-fold increased risk of endometriosis (OR 4.67, 95% CI 1.84–11.83, P = 0.001) and 3-fold increased risk of IVF failure (OR 3.33, 95% CI 1.38–8.03, P = 0.007). Our results demonstrate an association between ESR1 rs9340799 polymorphism and infertile women with endometriosis and also with women who were submitted to IVF procedures and had no blastocyst implantation.     

Validation and application of a novel integrated genetic screening method to a cohort of 1,112 men with idiopathic azoospermia or severe oligozoospermia

Microdeletions of the Y chromosome (YCMs), Klinefelter syndrome (47,XXY), and CFTR mutations are known genetic causes of severe male infertility, but the majority of cases remain idiopathic. Here, we describe a novel method using single molecule Molecular Inversion Probes (smMIPs), to screen infertile men for mutations and copy number variations affecting known disease genes. We designed a set of 4,525 smMIPs targeting the coding regions of causal (n = 6) and candidate (n = 101) male infertility genes. After extensive validation, we screened 1,112 idiopathic infertile men with non‐obstructive azoospermia or severe oligozoospermia. In addition to five chromosome YCMs and six other sex chromosomal anomalies, we identified five patients with rare recessive mutations in CFTR as well as a patient with a rare heterozygous frameshift mutation in SYCP3 that may be of clinical relevance. This results in a genetic diagnosis in 11–17 patients (1%–1.5%), a yield that may increase significantly when more genes are confidently linked to male infertility. In conclusion, we developed a flexible and scalable method to reliably detect genetic causes of male infertility. The assay consolidates the detection of different types of genetic variation while increasing the diagnostic yield and detection precision at the same or lower price compared with currently used methods.     

Defective recombination in infertile men

Two percent of men are infertile owing to defects in sperm production. In 10-15% of cases, Y chromosome deletions that encompass critical spermatogenesis genes are detected; in the remaining cases, the cause of infertility is unknown. In model organisms, defects in recombination genes cause infertility, germ cell aneuploidy and subsequent development of inviable or abnormal progeny. Several studies have also linked infertility and higher rates of germ cell aneuploidy in men and women. Thus, we reasoned that defective recombination may be a major cause of infertility in men with poor or no sperm production and we performed the first comparison of recombination parameters within populations of single spermatocytes from infertile and fertile men who reported for assisted reproduction. We observed that 10% of non-obstructive azoospermic men had significantly lower recombination frequencies than men with normal spermatogenesis. Furthermore, when we focused our analysis only on those men who had a pathological diagnosis of 'maturation arrest' due to arrest during sperm development, about half had detectable defects in recombination. In contrast, none of the men with normal spermatogenesis had defects in recombination. Thus, this study provides direct evidence that defects in recombination are linked to poor sperm production in a significant percentage of infertile men. Implications of this observation for the use of assisted reproductive technologies are especially relevant to consider, given that recombination is required to both introduce genetic variation and insure proper chromosome separation during meiosis.     

Factors associated with premature ovarian failure, early menopause and earlier onset of menopause in Japanese women

Objective

The purpose of this study is to clarify the median age at natural menopause and the proportions of women with premature ovarian failure (POF) and early menopause (EM) by using Kaplan–Meier cumulative estimates and differences in reproductive and lifestyle factors associated with POF, EM and median age at menopause in a large population of Japanese women.

Subjects and methods

This study is a cross-sectional analysis of the Japan Nurses’ Health Study (JNHS). We analyzed data for 24,152 pre- and postmenopausal women who were 40 years or older at the JNHS baseline survey.

Results

The overall estimated median age at natural menopause was 52.1 years, and the proportions of women with POF and EM were 0.28% and 1.67%, respectively. Older generation, cigarette smoking, low body mass index, regular menstruation cycles at 18–22 years of age, nulliparity and unilateral oophorectomy were associated with earlier onset of natural menopause.Only unilateral oophorectomy was associated with increased risk of POF, and nulliparity and unilateral oophorectomy were associated with increased risk of EM.

Conclusion

Unilateral oophorectomy is a common factor associated with earlier onset of menopause, EM and POF, although other reproductive and lifestyle factors are not associated with POF or EM.     

Genes Located In and Near the Human Pseudoautosomal Region are Located in the X-Y Pairing Region in Dog and Sheep

We cloned and mapped the dog and/or sheep homologues of two human pseudoautosomal genes CSF2RA and ANT3. We also cloned and mapped dog and/or sheep homologues of STS and PRKX, which are located nearby on the differential region of the human X and have related genes or pseudogenes on the Y. STS, as well as CSF2RA, mapped to the tips of the short arm of the sheep X and Y (Xp and Yp), and STS and PRKX, as well as ANT3, mapped to the tips of the dog Xp and Y long arm (Yq). These locations within the X-Y pairing regions suggest that the regions containing all these human Xp22.3-Xpter genes are pseudoautosomal in dog and sheep. This supports the hypothesis that a larger pseudoautosomal region (PAR) shared by eutherian groups was disrupted by chromosomal rearrangements during primate evolution. The absence of STS and ANT3 from the sex chromosomes in two prosimian lemur species must therefore represent a recent translocation from their ancestral PAR, rather than retention of a smaller ancestral PAR shared by mouse.     

Estrogen receptor alpha polymorphisms and fertility in populations with different reproductive patterns

The estrogen receptor (ER) plays an important role in mediating estrogen action on target tissues. ER-alpha, the most abundant, is found in all human reproductive tissues and studies on alpha-ER knockout mice have highlighted its role in reproduction. ER-alpha gene (ESR1) polymorphisms have been associated with a variety of disorders including human infertility. In this study, we examined the association of ESR1 PvuII and XbaI polymorphisms with fertility in two populations with different reproductive patterns and precisely in a sample of healthy Italian men and women (n=178) and in a sample of healthy African-Ecuadorian women (n=57). ESR1 xx and ppxx genotypes among the Italian men were found to be associated with an above-median number of children (P=0.01 and P=0.004, respectively). ESR1 pp genotype among the Italian women showed a tendency to be associated with a lower number of abortions (P=0.04), whereas ESR1 pp and ppxx genotypes among African-Ecuadorian women were associated with a higher number of children (P=0.02 and P=0.03, respectively). These results are consistent with previous observations indicating a role of ESR1 genotypes in human infertility and give insight into the complex interactions between genotypes and reproductive behaviours in human populations.     

Sensorineural deafness and male infertility: a contiguous gene deletion syndrome

Background

Syndromic hearing loss that results from contiguous gene deletions is uncommon. Deafness‐infertility syndrome (DIS) is caused by large contiguous gene deletions at 15q15.3.

Methods

Three families with a novel syndrome characterised by deafness and infertility are described. These three families do not share a common ancestor and do not share identical deletions. Linkage was established by completing a genome‐wide scan and candidate genes in the linked region were screened by direct sequencing.

Results

The deleted region is about 100 kb long and involves four genes (KIAA0377, CKMT1B, STRC and CATSPER2), each of which has a telomeric duplicate. This genomic architecture underlies the mechanism by which these deletions occur. CATSPER2 and STRC are expressed in the sperm and inner ear, respectively, consistent with the phenotype in persons homozygous for this deletion. A deletion of this region has been reported in one other family segregating male infertility and sensorineural deafness, although congenital dyserythropoietic anaemia type I (CDAI) was also present, presumably due to a second deletion in another genomic region.

Conclusion

We have identified three families segregating an autosomal recessive contiguous gene deletion syndrome characterised by deafness and sperm dysmotility. This new syndrome is caused by the deletion of contiguous genes at 15q15.3.     

Association Between Executive Dysfunction and Instrumental Activities of Daily Living: Racial and Ethnic Differences Among Community-Dwelling Older Adults in the Southeastern US.

ObjectiveExamining cultural differences in assessment of cognitive/functional disability among older Americans is needed. This analysis examined associations between day-to-day function, measured by activities of daily living (ADL), and cognition, measured by CLOX scores, among older African American (AA) and non-Hispanic White (nHW) community-dwelling women and men. Methods: Design- Cross-sectional.SettingHomes of community-dwelling older adults. Participants- 893 Medicare beneficiaries >65 living in west-central Alabama, without diagnoses of dementia, who were participants in the University of Alabama at Birmingham (UAB) Study of Aging, and who had complete data. Measurements- Physical function was assessed by self-reported ADL difficulty; cognitive function by CLOX, a clock drawing-task. Multivariable, linear regression models were used to examine associations within race/sex specific groups.ResultsAfter controlling for socio-demographic factors and comorbidities, CLOX1 scores were inversely and significantly correlated with ADL for AA men (β = ?0.205, P = 0.003). CLOX2 scores were similarly associated with ADL and IADL for the total group (β = ?0.118, P = 0.001, and β = ?0.180, P < 0.001, respectively); for ADL, significant associations were seen for AA men and nHW women (β = ?0.203, P = 0.004, and β = ?0.139, P = 0.02, respectively) and, for IADL, in AA women and men (β = ?0.156, P = 0.03, and β = ?0.24, P < 0.001, respectively).ConclusionWhile African American women reported the highest difficulty with ADLs and IADLs among all race/sex groups, CLOX1 scores were correlated with ADL for AA men only. CLOX1 may have limitations to identify functional disability for older AA women. [Word Count = 234].     

An SNP in protamine 1: a possible genetic cause of male infertility?

Gene targeting of the sperm nuclear proteins, the protamines, in mice leads to haploinsufficiency, abnormal chromatin compaction, sperm DNA damage, and male infertility. In order to investigate whether changes in amount or structure of the protamines could be a cause of human infertility, we sequenced the protamine genes of infertile men whose sperm appeared phenotypically similar to those of protamine deficient mice. We identified a heterozygous single nucleotide polymorphism (SNP) in the protamine (PRM1) gene in three infertile men (10% of the total infertile men analysed). This SNP disrupts one of the highly conserved arginine clusters needed for normal DNA binding. To rapidly screen for this SNP in infertile patients, we developed a simple PCR restriction fragment length polymorphism assay. This is the first report of a SNP in the PRM1 gene that appears associated with human male infertility.     

Chromosomal rearrangements in Xq and premature ovarian failure: mapping of 25 new cases and review of the literature

BACKGROUND: Chromosomal rearrangements in Xq are frequently associated with premature ovarian failure (POF) and have defined a POF 'critical region'. Search for genes responsible for the disorder has been elusive. METHODS: We report mapping of novel breakpoints of X;autosome-balanced translocations and interstitial deletions and a review of published X chromosome rearrangements. RESULTS: All the novel POF-associated rearrangements were mapped outside and often very distant from genes. The majority mapped to a gene-poor region in Xq21. In the same region, deletions were reported in women who apparently did not have problems conceiving. Expression analysis of genes flanking breakpoints clustered in a 2-Mb region of Xq21 failed to demonstrate ovary-specific genes. CONCLUSIONS: Our results excluded most of the possible explanations for the POF phenotype and suggested that POF should be ascribed to a position effect of the breakpoints on flanking genes. We also showed that while the X breakpoint may affect X-linked genes in the distal part of Xq, from Xq23 to Xq28, interruption of the critical region in Xq21 could be explained by a position effect of the Xq critical region on genes flanking the autosomal breakpoints.     

Absence of strong linkage disequilibrium between odorant receptor alleles and the major histocompatibility complex

The odorant receptor (OR) genes constitute the largest gene family among vertebrates. While over 800 loci are present in the human genome, their allele diversity is still poorly characterized. It has been hypothesized that the products of OR genes can be relevant in the reproductive context, thereby interacting with products of genes of the major histocompatibility complex (MHC). Here we investigated the genetic diversity of the OR2W6P, OR2B8P, OR1F12 and OR12D2 genes, in order to define haplotypes and haplotype frequencies. We measured levels of linkage disequilibrium (LD) between these OR genes and the MHC genes HLA-A, HLA-B and HLA-DRB1. This was accomplished through the assessment of 30 single nucleotide polymorphisms (SNPs) in samples from 61 family trios. We characterized 26 alleles among the four OR genes and identified three SNPs that had not yet been reported. Based on our haplotype analysis, LD spanning the OR-HLA region is not very strong, and therefore not enough to enable selection regarding specific HLA-OR haplotypes.     

Associations of Y-chromosome subdeletion gr/gr with the prevalence of Y-chromosome haplogroups in infertile patients

Microdeletions in azoospermia factor (AZF) region on distal Yq are associated with male infertility and spermatogenic failure due to intra-chromosomal homologous recombination between large nearly identical repeat amplicons and are found in ∼10% of azoospermic and severe oligozoospermic cases. Although AZFc is deleted in azoospermia or oligozoospermia, no definitive conclusion has been drawn for the role of partial AZFc deletions to spermatogenic failure. Therefore, this study is planned to investigate the role of gr/gr subdeletions in individuals with spermatogenic failure and to find its relationship with Y chromosome haplogroups (HGs) in infertile men from Indian population. It is a case-control study involving 236 azoospermic, 182 oligospermic and 240 healthy normozoospermic men. We found 18 gr/gr, 11 b1/b3 and 2 b2/b3 subdeletions in azoospermic patients and 12 gr/gr, 5 b1/b3 and 4 b2/b3 subdeletions in oligospermic patients. However, we also found seven gr/gr deletions in normozoospermic men. Seven patients each with spermatogenic arrest and oligospermia who carry gr/gr subdeletions have deleted DAZ3/DAZ4 genes. A total of 11 patients with sertoli cell-only syndrome (SCOS) and 5 oligospermic patients with gr/gr subdeletions also have DAZ1/DAZ2 genes deleted indicating that deletions of DAZ genes contributed differently to damage to spermatogenic process. L1 HG is found in patients showing b1/b3 subdeletions, whereas HG H1a2 and H1b were found in normozoospermic individuals with gr/gr subdeletions. Our results provide evidence of association between the occurrence of subdeletions and male infertility as well as the severity of the spermatogenic failure.     

The sexual dimorphism of anticardiolipin autoantibodies in acute Q fever patients

ObjectivesQ fever is a zoonotic disease caused by Coxiella burnetii which affects men more than women (sex ratio men/women: 2.2). Acute Q fever complications are associated with elevation of anticardiolipin (aCL) antibodies. Here, we investigate the sexual dimorphism of aCL antibodies during acute C. burnetii infection.MethodsIgG aCL antibodies were evaluated at the time of Q fever serological diagnosis with enzyme-linked immunosorbent assay. Results were analysed according to sex.ResultsAmong the 1323 patients with Q fever tested for aCL, 1013 had acute Q fever (692 men/321 women) and 310 had persistent focalized infection (226 men/84 women). In cases of acute Q fever, men presented a significantly higher proportion of positive aCL antibodies (351/692, 50.7%) than women (113/321, 35.2%) (p <0.05). In addition, men had significantly higher aCL antibodies levels than women (p <0.001).ConclusionsWe highlight a relationship between sex and markers of autoimmunity during Q fever. Further investigations are necessary to better understand the mechanisms of this sexual dimorphism.