Molecular definition of Xq common-deleted region in patients affected by premature ovarian failure

High-resolution cytogenetic analysis of a large number of women with premature ovarian failure (POF) identified six patients carrying different Xq chromosome rearrangements. The patients (one familial and five sporadic cases) were negative for Turner's stigmata and experienced a variable onset of menopause. Microsatellite analysis and fluorescent in situ hybridization (FISH) were used to define the origin and precise extension of the Xq anomalies. All of the patients had a Xq chromosome deletion as the common chromosomal abnormality, which was the only event in three cases and was associated with partial Xp or 9p trisomies in the remaining three. Two of the Xq chromosome deletions were terminal with breakpoints at Xq26.2 and Xq21.2, and one interstitial with breakpoints at Xq23 and Xq28. In all three cases, the del(X)s retained Xp and Xq specific telomeric sequences. One patient carries a psu dic(X) with the deletion at Xq22.2 or Xq22.3; the other two [carrying (X;X) and (X;9) unbalanced translocations, respectively] showed terminal deletions with the breakpoint at Xq22 within the DIAPH2 gene. Furthermore, the rearranged X chromosomes were almost totally inactivated, and the extent of the Xq deletions did not correlate with the timing of POF. In agreement with previous results, these findings suggest that the deletion of a restricted Xq region may be responsible for the POF phenotype. Our analysis indicates that this region extends from approximately Xq26.2 (between markers DXS8074 and HIGMI) to Xq28 (between markers DXS 1113 and ALD) and covers approximately 22 Mb of DNA. These data may provide a starting point for the identification of the gene(s) responsible for ovarian development and folliculogenesis.     

Physical mapping of nine Xq translocation breakpoints and identification of XPNPEP2 as a premature ovarian failure candidate gene

Women with balanced translocations between the long arm of the X chromosome (Xq) and an autosome frequently suffer premature ovarian failure (POF). Two "critical regions" for POF which extend from Xq13-->q22 and from Xq22-->q26 have been identified using cytogenetics. To gain insight into the mechanism(s) responsible for ovarian failure in women with X;autosome translocations, we have molecularly characterized the translocation breakpoints of nine X chromosomes. We mapped the breakpoints using somatic cell hybrids retaining the derivative autosome and densely spaced markers from the X-chromosome physical map. One of the POF-associated breakpoints in a critical region (Xq25) mapped to a sequenced PAC clone. The translocation disrupts XPNPEP2, which encodes an Xaa-Pro aminopeptidase that hydrolyzes N-terminal Xaa-Pro bonds. XPNPEP2 mRNA was detected in fibroblasts that carry the translocation, suggesting that this gene at least partially escapes X inactivation. Although the physiologic substrates for the enzyme are not known, XPNPEP2 is a candidate gene for POF. Our breakpoint mapping data will help to identify additional candidate POF genes and to delineate the Xq POF critical region(s).     

X-linked premature ovarian failure: a complex disease

Involvement of the X chromosome in premature ovarian failure was demonstrated by the relatively frequent chromosomal rearrangements in patients, but the requirement of two X chromosomes for ovarian function was quite unexplained until recently. Review of the data on chromosomal rearrangements suggests that several genes along the X chromosomes contribute to ovarian function. In most instances, no single X chromosome gene has a causative role in premature ovarian failure, and the phenotype is likely to derive from the additive effect of X-linked and non-X-linked factors. Recent data on a small group of balanced X-autosome translocations showed that X-linked premature ovarian failure might also be caused by a different mechanism, namely position effect of the X chromosome on non-X-linked genes, and suggest a peculiar organization of the X chromosome during oogenesis.     

Cytogenetic study of mentally retarded children in Taipei

560 blood samples collected from mentally retarded children in Taipei were karyotypically analyzed for the incidence of fragile X and other chromosome abnormalities. The fragile site at Xq27.3 was observed in 18 patients (3.21%), 11 males and 7 females, out of the 560 blood cultures using M medium. Down syndrome (6.25%), 24 males and 11 females, was the other major category of abnormality. Other abnormalities, including inversion, translocation, deletion, duplication, ring as well as an extra marker chromosome were observed. The overall incidence of chromosomal abnormalities in these children was 14.82%.     

Genetic aspects of premature ovarian failure

Among the causes of premature ovarian failure (POF) two groups of factors are reported: factors which lead to decrease of follicular number and factors which stimulate follicular atresia. In the first group genetic factors are the most important whereas in the second: enzymatic autoimmunological, iatrogenic, toxins and infections are reported. In 1986 familiar POF on the background of long arm of chromosome X deletion was reported. Other chromosomes which are important for normal ovarian function are: chromosome 21 (AIRE gene), chromosome 11 (gene of beta FSH, ATM gene), chromosome 3 (gene responsible for BEPS syndrome) and chromosome 2 (genes of FSH and LH receptors). In this review the role of these genes and results of several epidemiological studies are reported.     

Transcription factor FIGLA is mutated in patients with premature ovarian failure

Premature Ovarian Failure (POF) is a genetically heterogenous disorder that leads to hypergonadotropic ovarian failure and infertility. We screened 100 Chinese women with POF for mutations in the oocyte-specific gene FIGLA and identified three variants in four women: missense mutation c.11C --> A (p.A4E) was found in two women; deletion c. 15-36 del (p.G6fsX66), resulting in a frameshift that leads to haploinsufficiency, was found in one woman; and deletion c.419-421 delACA (p.140 delN) was found in one. Functional analyses by the yeast two-hybrid assay demonstrated that the p.140 delN mutation disrupted FIGLA binding to the TCF3 helix-loop-helix (HLH) domain. Our findings show that a subset of Chinese women with sporadic, premature ovarian failure harbor mutations in FIGLA.     

Imprinting effect in premature ovarian failure confined to paternally inherited fragile X premutations

Fragile X premutations are considered to be a risk factor for premature ovarian failure (POF), which is usually defined as menopause at age <40 years. Since premutations may be inherited from either the mother or the father, we evaluated the influence of the inheritance pattern on the duration of reproductive life in female carriers. The occurrence of POF and age at menopause in women with a paternally inherited fragile X premutation (PIP) were compared to those in women with a maternally inherited fragile X premutation (MIP). We identified 148 women in whom the parental origin of the premutation could be determined. In 109 of these women we were able to establish whether POF had occurred: 82 women had a PIP, and 27 had a MIP. Twenty-three of the women (28%) with a PIP had POF, versus only 1 (3.7%) with a MIP (two -tailed Fisher's exact test; P=. 007). Kaplan-Meier analysis of all 148 premutations showed that the age at menopause was significantly lower in the women with a PIP than in the woman with a MIP (Breslow test in Kaplan-Meier analysis; P=.003). Our data strongly suggest that, when POF occurs in fragile X premutation carriers, a considerable proportion of the premutations are inherited paternally (parent-of-origin effect). We hypothesize that this may be owing to a paternal genomic imprinting effect.     

Epigenetic control of the critical region for premature ovarian failure on autosomal genes translocated to the X chromosome: a hypothesis

Chromosomal rearrangements in Xq are frequently associated to premature ovarian failure (POF) and have contributed to define a POF “critical region” from Xq13.3 to Xq26. Search for X-linked genes responsible for the phenotype has been elusive as most rearrangements did not interrupt genes and many were mapped to gene deserts. We now report that ovary-expressed genes flanked autosomal breakpoints in four POF cases analyzed whose X chromosome breakpoints interrupted a gene poor region in Xq21, where no ovary-expressed candidate genes could be found. We also show that the global down regulation in the oocyte and up regulation in the ovary of X-linked genes compared to the autosomes is mainly due to genes in the POF “critical region”. We thus propose that POF, in X;autosome balanced translocations, may not only be caused by haploinsufficiency, but also by a oocyte-specific position effect on autosomal genes, dependent on dosage compensation mechanisms operating on the active X chromosome in mammals.     

Molecular genetic and cytogenetic characterization of a partial Xp duplication and Xq deletion in a patient with premature ovarian failure

Background

The etiology of premature ovarian failure (POF) still remains undefined. Although the majority of clinical cases are idiopathic, there are possibilities of the underestimation of the most common etiologies, probably genetic causes. By reporting a case of POF with a partial Xp duplication and Xq deletion in spite of a cytogenetically 46,XX normal karyotype, we look forward that the genetic cause of POF will be investigated more methodically.

Methods

We performed a basic and clinical study at a university hospital-affiliated fertility center. The study population was a POF patient and her family. Cytogenetic analysis, FMR1 gene analysis, multiplex ligation-dependent probe amplification (MLPA), fluorescent in situ hybridization (FISH), and oligonucleotide-array based comparative genomic hybridization (array CGH) were performed.

Results

In spite of normal cytogenetic analysis in the proband and her mother and younger sister, FMR1 gene was not detected in the proband and her younger sister. In Southern blot analysis, the mother showed a normal female band pattern, but the proband and her younger sister showed no 5.2 kb methylated band. The abnormal X chromosome of the proband and her sister was generated from the recombination of an inverted X chromosome of the mother during maternal meiosis, and the karyotype of the proband was 46,XX,rec(X)dup(Xp)inv(X)(p22.1q27.3).

Conclusion

Array CGH followed by FISH allowed precise characterization of the der(X) chromosome and the initial karyotype of the proband had been changed to 46,XX,rec(X)dup(Xp)inv(X)(p22.3q27.3)mat.arr Xp22.33p22.31(216519–8923527)x3,Xq27.3q28(144986425–154881514)x1. This study suggests that further genetic investigation may be needed in the cases of POF with a cytogenetically 46,XX normal karyotype to find out the cause and solution for these disease entities.     

The use of DNA analysis for diagnostics of hereditary premature ovarian failure

Methods of DNA-analysis of 769G --> A mutations in INHalpha1 gene and CGG-repeats polymorphism in FMRI gene have been developed for creating test-systems for genetically caused forms of premature ovarian failure (POF) diagnostics. The frequency of 769G --> A mutation among women population in Ukraine was established and, by preliminary calculations, makes up 2.8%. Results of analysis of CGG-repeats numbers in FMRI gene in the group of 215 women (oocyte donors) revealed five persons with CGG-repeats numbers, that exceeds the normal one (42 copies). Thus the frequency of persons with allels with high risk of premutation in FMRI gene is 2.3%. The results of our research confirm the actuality of genetic tests of mutations in INHalpha1 and FMR1 genes among the women of reproductive age with the purpose of POF prognosis and prevention the birth of children with fragile X syndrome.     

Disruption of POF1B binding to nonmuscle actin filaments is associated with premature ovarian failure

Premature ovarian failure (POF) is characterized by elevated gonadotropins and amenorrhea in women aged <40 years. In a Lebanese family with five sisters who received the diagnosis of POF, we established linkage to the long arm of the X chromosome (between Xq21.1 and Xq21.3.3), using whole-genome SNP typing and homozygosity-by-descent mapping. By sequencing one candidate gene within that region, POF1B, we identified a point mutation localized in exon 10. This substitution of a nucleotide (G-->A), at position 1123, results in an arginine-->glutamine mutation of the protein sequence at position 329 (mutation R329Q). All the affected family members were homozygous for the mutation, whereas the unaffected members were heterozygous. Because POF1B shares high homology with the tail portion of the human myosin, we assessed the ability of both wild-type and mutant POF1B proteins to bind nonmuscle actin filaments in vitro. We found that the capacity of the mutant protein to bind nonmuscle actin filaments was diminished fourfold compared with the wild type, suggesting a function of POF1B in germ-cell division. Our study suggests that a homozygous point mutation in POF1B influences the pathogenesis of POF by altering POF1B binding to nonmuscle actin filaments.     

Primary amenorrhoea in a patient with mosaicism for monosomy X and a derivative X-chromosome

Primary amenorrhoea is defined as the absence of menstruation in phenotypic women aged 16 years or older, if secondary sexual characteristics are present. X chromosome abnormalities probably comprise about one half of all cases, including Turner syndrome and X chromosome rearrangements. Conventional banding cytogenetic methods might miss the accurate detection of structural chromosome abnormalities. The fluorescence in situ hybridization (FISH) and multicolor FISH techniques are required to interpret specific chromosomal rearrangement. As far as we know, we report the first case with chromosome mosaicism for monosomy X and terminal deletion of Xq26 with duplication of Xp11-->pter. In spite of the fact that a 45,X karyotype was detected in 46% of lymphocytes, she was tall and her secondary sexual characteristics were moderately developed, including breast, pubic and axillary hair stages. Cytogenetic and FISH analyses should be considered for patients presenting primary amenorrhoea even if there are no other clinical features suggestive of chromosome abnormality.     

Blood Cell Mitochondrial DNA Content and Premature Ovarian Aging

Primary ovarian insufficiency (POI) is a critical fertility defect characterized by an anticipated and silent impairment of the follicular reserve, but its pathogenesis is largely unexplained. The frequent maternal inheritance of POI together with a remarkable dependence of ovarian folliculogenesis upon mitochondrial biogenesis and bioenergetics suggested the possible involvement of a generalized mitochondrial defect. Here, we verified the existence of a significant correlation between blood and ovarian mitochondrial DNA (mtDNA) content in a group of women undergoing ovarian hyperstimulation (OH), and then aimed to verify whether mtDNA content was significantly altered in the blood cells of POI women. We recruited 101 women with an impaired ovarian reserve: 59 women with premature ovarian failure (POF) and 42 poor responders (PR) to OH. A Taqman copy number assay revealed a significant mtDNA depletion (P<0.001) in both POF and PR women in comparison with 43 women of similar age and intact ovarian reserve, or 53 very old women with a previous physiological menopause. No pathogenic variations in the mitochondrial DNA polymerase γ (POLG) gene were detected in 57 POF or PR women with low blood mtDNA content. In conclusion, blood cell mtDNA depletion is a frequent finding among women with premature ovarian aging, suggesting that a still undetermined but generalized mitochondrial defect may frequently predispose to POI which could then be considered a form of anticipated aging in which the ovarian defect may represent the first manifestation. The determination of mtDNA content in blood may become an useful tool for the POI risk prediction.     

Comparative genomic hybridization in combination with flow cytometry improves results of cytogenetic analysis of spontaneous abortions

More than 50% of spontaneous abortions (SAs) have abnormal chromosomes; the most common abnormalities are trisomy, sex chromosome monosomy, and polyploidy. Conventional cytogenetic analysis of SAs depends on tissue culturing and is associated with a significant tissue culture failure rate and contamination by maternally derived cells. Comparative genomic hybridization (CGH), in combination with flow cytometry (FCM), can detect numerical and unbalanced structural chromosomal abnormalities associated with SAs while avoiding the technical problems associated with tissue culture. Routine cytogenetic and CGH analysis was performed independently on tissue from 301 SAs. Samples shown to be chromosomally balanced by CGH were analyzed by FCM to determine ploidy. Of 253 samples successfully analyzed by both approaches, there was an absolute correlation of results in 235 (92.8%). Of the 18 cases with discrepancies between cytogenetic and CGH/FCM results, an explanation could be found in 17. Twelve samples produced a 46,XX karyotype by cytogenetics, whereas CGH/FCM demonstrated aneuploidy/polyploidy or a male genome, indicating maternal contamination of the tissue cultures. In two cases, where tetraploidy was demonstrated by cytogenetics and diploidy by FCM, tissue culture artifact is implied. In three cases, CGH demonstrated an aneuploidy, and cytogenetics demonstrated hypertriploidy. In one unexplainable case, aneuploidy demonstrated by CGH could not be detected by repeat CGH analysis, conventional cytogenetic, or FISH analysis. These results demonstrate that CGH supplemented with FCM can readily identify chromosomal abnormalities associated with SAs and, by avoiding maternal contamination and tissue culture artifacts, can do so with a lower failure rate and more accuracy than conventional cytogenetic analysis.     

Human placenta‐derived mesenchymal stem cells inhibit apoptosis of granulosa cells induced by IRE1α pathway in autoimmune POF mice

Previous studies have shown that the ovarian failure in autoimmune‐induced premature ovarian failure (POF) mice could be improved by the transplantation of human placenta‐derived mesenchymal stem cells (hPMSCs); however, the protective mechanism of hPMSCs transplantation on ovarian dysfunction remains unclear. Ovarian dysfunction is closely related to the apoptosis of granulosa cells (GCs). To determine the effects of hPMSCs transplantation on GCs apoptosis, an autoimmune POF mice model was established with zona pellucida glycoprotein 3 (ZP3) peptide. It is reported that the inositol‐requiring enzyme 1α (IRE1α) and its downstream molecules play a central role in the endoplasmic reticulum (ER) stress‐induced apoptosis pathway. So the aim of this study is to investigate whether hPMSCs transplantation attenuated GCs apoptosis via inhibiting ER stress IRE1α signaling pathway. The ovarian dysfunction, follicular dysplasia, and GCs apoptosis were observed in the POF mice. And the IRE1α pathway was activated in ovaries of POF mice, as demonstrated by, increased X‐box binding protein 1 (XBP1), up‐regulated 78 kDa glucose‐regulated protein (GRP78) and caspase‐12. Following transplantation of hPMSCs, the ovarian structure and function were significantly improved in POF mice. In addition, the GCs apoptosis was obviously attenuated and IRE1α pathway was significantly inhibited. Transplantation of hPMSCs suppressed GCs apoptosis‐induced by ER stress IRE1α signaling pathway in POF mice, which might contribute to the hPMSCs transplantation‐mediating ovarian function recovery.     

Chromosomal evaluation in a group of Tunisian patients with non-obstructive azoospermia and severe oligozoospermia attending a Tunisian cytogenetic department

Male infertility is the cause in half of all childless partnerships. Numerous factors contribute to male infertility, including chromosomal aberrations and gene defects. Few data exist regarding the association of these chromosomal aberrations with male infertility in Arab and North African populations. We therefore aimed to evaluate the frequency of chromosomal aberrations in a sample of 476 infertile men with non-obstructive azoospermia (n = 328) or severe oligozoospermia (n = 148) referred for routine cytogenetic analysis to the department of cytogenetics of the Pasteur Institute of Tunis. The overall incidence of chromosomal abnormalities was about 10.9%. Out of the 52 patients with abnormal cytogenetic findings, sex chromosome abnormalities were observed in 42 (80.7%) including Klinefelter syndrome in 37 (71%). Structural chromosome abnormalities involving autosomes (19.2%) and sex chromosomes were detected in 11 infertile men. Abnormal findings were more prevalent in the azoospermia group (14.02%) than in the severe oligozoospermia group (4.05%). The high frequency of chromosomal alterations in our series highlights the need for efficient genetic testing in infertile men, as results may help to determine the prognosis, as well as the choice of an assisted reproduction technique. Moreover, a genetic investigation could minimize the risk of transmitting genetic abnormalities to future generations.     

Study of FMR1 gene association with ovarian dysfunction in a sample from the Basque Country

Premature ovarian failure (POF) is defined as cessation of menses before the age of 40. The most significant single gene associated with POF is the Fragile X Mental Retardation 1 gene (FMR1). In the present work we screened women with fertility problems from the Basque Country in order to determine, whether in these women, FMR1 CGG repeat size in the intermediate and premutation range was associated with their pathology, and whether intermediate and premutation carriers had endocrine signs of diminished ovarian function, using the most established measure of ovarian reserve, the gonadotropin FSH. A patient sample of 41 women with ovarian insufficiency and a control sample of 32 women with no fertility problems from the Basque Country were examined. The patient sample was classified into three categories according to the results of the retrospective assessment of their ovarian function. In group 2 of patients, women with irregular cycles, reduced fecundity and FSH levels ≥ 10 IU/l, there is a significant increase in the number of intermediate and premutation FMR1 alleles (35–54 CGG repeats). In group 3 of patients, women with amenorrhea for at least four consecutive months and FSH levels ≥ 10 IU/l, a significant increase in the number of intermediate FMR1 alleles (35–54 CGG repeats) was found in patients compared with controls. In this group all the patients had a serum concentration > 40 IU/l. The results suggest that in the analysed Basque sample the FMR1 gene has a role in the aetiology of POF. However, elevated FSH levels are more related to the menstrual cycle pattern than to the CGG repeat size.     

Complete response to FOLFOX4 therapy in a patient with advanced urothelial cancer: a case report

The ring chromosome is a circular, structural abnormality composed of either multiple chromosomes or a single chromosome with loss of genetic material at one or both ends. This chromosomal rearrangement is often unstable with frequent recombinations and may be accompanied by either loss or amplification of genetic material[1]. Considering that ring chromosomes are rare in acute myelogenous leukemia (AML), it is difficult to risk stratify patient prognosis, particularly when the ring chromosome occurs as the sole abnormality. Here we report a case of a ring chromosome 18 abnormality in a patient with newly diagnosed AML with monocytic differentiation. Cytogenetic analysis demonstrated 46, XY, r(18)(p11q21) karyotype in 19 of 34 evaluated metaphase cells. The patient received induction chemotherapy and subsequent allogeneic cord blood transplant from a sex-matched donor, and remained in hematologic and cytogenetic remission for 120 days post transplant. Soon after, he developed post transplant lymphoproliferative disorder and died of multi-organ failure. Although r(18) chromosomal abnormalities were not classified in the recent updated evidence-and expert opinion-based recommendations for the diagnosis and management of AML (likely due to the small number of reported cases), the patient was treated as high risk with stem cell transplantation. This was based on the unstable nature of the ring chromosome and the poor outcomes described in the literature of patients with sole ring 18 abnormalities.     

Reactive oxygen species‐induced SIAH1 promotes granulosa cells' senescence in premature ovarian failure

Reactive oxygen species (ROS) exposure triggers granulosa cells'' (GCs) senescence, which is an important causal factor for premature ovarian failure (POF). However, underlying mechanism in this process remains unknown. In our study, we observed increased ROS levels in POF ovarian tissues, POF patient follicular GCs and cyclophosphamide (CTX) pretreated GCs. Correspondingly, increased SIAH1, reduced TRF2 and GC senescence were also found in these cases. Silencing of SIAH1 rescued ROS‐induced TRF2 reduction and cell senescence in GCs. Moreover, SIAH1 co‐localized with TRF2 in the cytoplasm, facilitating its ubiquitination degradation, further leading to telomere abnormalities in GCs. In conclusion, our findings indicate that ROS induces telomere abnormalities by augmenting SIAH1‐mediated TRF2 degradation, leading to cell senescence in GCs in POF processing.