A non-sense MCM9 mutation in a familial case of primary ovarian insufficiency.

Primary ovarian insufficiency (POI) results in an early loss of ovarian function, and remains idiopathic in about 80% of cases. Here, we have performed a complete genetic study of a consanguineous family with two POI cases. Linkage analysis and homozygosity mapping identified 12 homozygous regions with linkage, totalling 84 Mb. Whole-exome sequencing of the two patients and a non-affected sister allowed us to detect a homozygous causal variant in the MCM9 gene. The variant c.1483G>T [p.E495*], confirmed using Sanger sequencing, introduced a premature stop codon in coding exon 8 and is expected to lead to the loss of a functional protein. MCM9 belongs to a complex required for DNA repair by homologous recombination, and its impairment in mouse is known to induce meiotic recombination defects and oocyte degeneration. A previous study recently described two consanguineous families in which homozygous mutations of MCM9 were responsible for POI and short stature. Interestingly, the affected sisters in the family described here had a normal height. Altogether, our results provide the confirmation of the implication of MCM9 variants in POI and expand their phenotypic spectrum.

A 11.7-kb deletion triggers intersexuality and polledness in goats.

Mammalian sex determination is governed by the presence of the sex determining region Y gene (SRY) on the Y chromosome. Familial cases of SRY-negative XX sex reversal are rare in humans, often hampering the discovery of new sex-determining genes. The mouse model is also insufficient to correctly apprehend the sex-determination cascade, as the human pathway is much more sensitive to gene dosage. Other species might therefore be considered in this respect. In goats, the polled intersex syndrome (PIS) mutation associates polledness and intersexuality. The sex reversal affects exclusively the XX individuals in a recessive manner, whereas the absence of horns is dominant in both sexes. The syndrome is caused by an autosomal gene located at chromosome band 1q43 (ref. 9), shown to be homologous to human chromosome band 3q23 (ref. 10). Through a positional cloning approach, we demonstrate that the mutation underlying PIS is the deletion of a critical 11.7-kb DNA element containing mainly repetitive sequences. This deletion affects the transcription of at least two genes: PISRT1, encoding a 1.5-kb mRNA devoid of open reading frame (ORF), and FOXL2, recently shown to be responsible for blepharophimosis ptosis epicanthus inversus syndrome (BPES) in humans. These two genes are located 20 and 200 kb telomeric from the deletion, respectively.

Donor splice-site mutations in WT1 are responsible for Frasier syndrome.

Frasier syndrome (FS) is a rare disease defined by male pseudo-hermaphroditism and progressive glomerulopathy. Patients present with normal female external genitalia, streak gonads and XY karyotype and frequently develop gonadoblastoma. Glomerular symptoms consist of childhood proteinuria and nephrotic syndrome, characterized by unspecific focal and segmental glomerular sclerosis, progressing to end-stage renal failure in adolescence or early adulthood. No case of Wilms' tumour has been reported, even in patients with extended follow-up. In contrast with FS patients, most individuals with Denys-Drash syndrome (DDS; refs 6,7) have ambiguous genitalia or a female phenotype, an XY karyotype and dysgenetic gonads. Renal symptoms are characterized by diffuse mesangial sclerosis, usually before the age of one year, and patients frequently develop Wilms' tumour. Mutations of the Wilms'-tumour gene, WT1, cause different pathologies of the urogenital system, including DDS. WT1 is composed of ten exons and encodes a protein with four zinc-finger motifs and transcriptional and tumour-suppressor activities. Alternative splicing generates four isoforms: the fifth exon may or may not be present, and an alternative splice site in intron 9 allows the addition of three amino acids (KTS) between the third and fourth zinc fingers of WT1 (ref. 17). Here we demonstrate that FS is caused by mutations in the donor splice site in intron 9 of WT1, with the predicted loss of the +KTS isoform. Examination of WT1 transcripts indeed showed a diminution of the +KTS/-KTS isoform ratio in patients with FS.

Genetic counseling for the orthodox jewish couple undergoing preimplantation genetic diagnosis.

Orthodox Jewish patients who seek genetic counseling are often placed in a difficult position of having to choose between their desire to follow Jewish religious instruction (halacha) and following the advice of the genetic counselor. In this article we will present the work of the Puah Institute based in Jerusalem that is dedicated to assisting and guiding such couples to navigate through the medical system and medical recommendations and create a harmony between modern genetic counseling and the Orthodox Jewish tradition. In light of the expanding use of preimplantation genetic diagnosis (PGD) for a variety of medical and non-medical conditions, this dilemma is even more poignant. There is an ethical debate regarding PGD and the correct parameters for its use. Here we present the Orthodox Jewish view of the use and abuse of PGD. We present three case studies that sought the assistance and guidance of the Puah Institute. Each of these cases raises ethical dilemmas for the genetic counselor and for the rabbinic counselor. We discuss; the status of the embryo, the status of a carrier of a genetic abnormality and whether PGD is an obligation or good practice. In addition we deal with whether PGD and the search for the desired traits can be defined as eugenics or not.

Foxl-2 in gonad development and pathology.

The Foxl-2 gene is involved in eyelid and ovary development. Mutations can lead to a shortened protein and malformations such as BPES associated or not to POF. Forkhead point mutation C134W is a marker of adult type granulosa cell tumors only. Foxl-2 dysregulation is also present in DSD and DSD associated tumors such as Gonadoblastoma and gonadoblastoma like intratubular undetermined germ cell neoplasia. A similar spectrum of pathology involvement is also found for WT1 and RET and gives a new insight into the relationship between development, malformations and oncogenesis.

Absence of allogeneic restriction in human T-cell-mediated cytotoxicity to Epstein-Barr virus-infected target cells. Demonstration of an HLA-linked control at the effector level.

Peripheral T lymphocytes from patients with infectious mononucleosis (IM) are sensitized in vivo against the Epstein-Barr virus (EBV). The expression of HLA-A, B, or C molecules at the target cell surface is necessary for the cytotoxic reaction because (a) EBV-positive Daudi cells lacking HLA-A, B, and C determinants are resistant to anti-EBV T-cell lysis, (b) cytolysis of EBV-positive target cells can be consistently inhibited by anti-HLA-A, B, and C and anti-beta 2 microglobulin antibodies. However, no evidence for allogeneic restriction in this system was apparent as (a) cytotoxic T lymphocytes (CTL) from one given individual could exert a cytotoxicity of a similar magnitude on different EBV-positive target cells, regardless of the number of HLA-A or B specificities shared by the effectors and targets; (b) CTL from IM patients were able to kill target cells without any HLA-A or B antigen in common; and (c) T5-1 variants lacking one or two HLA antigens at the A, B, or D locus are killed to the same extent as the parental cells. 7 of the 9 IM patients with detectable circulating anti-EBV CTL carried the HLA-A1 antigen, whereas none of the 16 IM patients lacking detectable peripheral CTL were HLA-A1 positive (mean specific lysis of T5-1 target cells by T cells from HLA-A1 positive patients: 29.3 vs. 0.6% in HLA-A1-negative patients) (P less than 10(-9)). These data suggest an HLA-A1-linked gene control of the magnitude of the anti-EBV CTL response. Thus, the HLA region appears to act at two different level sin the T-cell-mediated lysis of EBV-infected cells by controlling first, the development of anti-EBV and second, the expression of HLA-A, B, and C molecules involved as recognition structures at the target cell surface.

Association of the H-Y male antigen with beta2-microglobulin on human lymphoid and differentiated mouse teratocarcinoma cell lines.

The expression of the H-Y antigen has been tested on several human lymphoid lines and mouse teratocarcinoma cell lines during differentiation. The human male lymphoid cell line Raji is a very useful target for studies of the H-Y antigen by lymphocytotoxicity test with rat anti-H-Y sera. With a few exceptions, all cells carrying the Y chromosome were H-Y positive. One of the exceptions is the human Daudi cell line which, besides lacking H-Y antigen, also lacks beta2-microglobulin. We have studied a possible association between the H-Y antigen, beta2-microglobulin, and HLA antigen with redistribution experiments. The results strongly suggest that H-Y antigen is not associated with HLA antigens but with beta2-microglobulin.

Functional evidence implicating FOXL2 in non-syndromic premature ovarian failure and in the regulation of the transcription factor OSR2.

BACKGROUND: FOXL2 encodes a forkhead transcription factor whose mutations are responsible for the blepharophimosis-ptosis-epicanthus inversus syndrome (BPES), involving craniofacial/palpebral abnormalities often associated with premature ovarian failure (POF). RESULTS: We describe a FOXL2 variant (p.Gly187Asp) in a case of POF without BPES. The subcellular localisation of FOXL2-G187D was normal but its transactivation capacity tested on two reporter promoters, one of which should be relevant to the ovary, was significantly lower than that of normal FOXL2. However, FOXL2-G187D was able to activate strongly a reporter construct driven by the promoter of Osr2 (odd-skipped related 2 transcription factor), which we have suggested to be a crucial target of FOXL2 in the craniofacial region. This is compatible with the absence of BPES in our patient. CONCLUSIONS: Our data provide evidence in favour of the implication of FOXL2 variants in non-syndromic POF and confirm the regulatory interaction between FOXL2 and OSR2 whose perturbation might contribute to the palpebral abnormalities observed in BPES patients.

Interferon alpha inhibits a Src-mediated pathway necessary for Shigella-induced cytoskeletal rearrangements in epithelial cells.

Shigella flexneri, the causative agent of bacillary dysentery, has the ability to enter nonphagocytic cells. The interferon (IFN) family of cytokines was found to inhibit Shigella invasion of cultured epithelial cells. We show here that IFN-alpha inhibits a Src-dependent signaling cascade triggered by Shigella that leads to the reorganization of the host cell cytoskeleton. Immunofluorescence studies showed that IFN-alpha inhibits Shigella-induced actin polymerization required for bacterial entry into cells. Phosphorylation of cortactin, a Src-substrate specifically tyrosyl-phosphorylated during Shigella entry, was inhibited by IFN-alpha. Overexpression of a dominant interfering form of pp60c-src led to inhibition of Shigella-induced cytoskeletal rearrangements and decreased cortactin phosphorylation indicating a role for Src in Shigella entry. Also, Shigella uptake in cells that expressed constitutively active Src was unaffected by IFN-alpha treatment. We conclude that Src kinase activity is necessary for Shigella invasion of epithelial cells and that IFN-alpha inhibits this Src-dependent signaling pathway.

A glycolipid antigen associated with Burkitt lymphoma defined by a monoclonal antibody.

The antigen defined by a rat monoclonal antibody directed to a Burkitt lymphoma cell line was identified as globotriaosylceramide [Gal alpha (1 leads to 4)-Gal beta (1 leads to 4)-Glc beta (1 leads to 1)-ceramide]. The antibody demonstrated a strict steric specificity since it did not react with globoisotriaosylceramide [Gal alpha (1 leads to 3)-Gal beta (1 leads to 4)-Glc beta (1 leads to 1)-ceramide], the positional isomer of the antigen associated with the Burkitt lymphoma. Chemical analysis of various Burkitt lymphoma cell lines revealed that the Burkitt lymphoma cells contained more than 100 times as much of the glycolipid antigen as was found in other human lymphoma and leukemia cell lines.

A human Y-linked DNA polymorphism and its potential for estimating genetic and evolutionary distance.

A human DNA sequence (p12f2), derived from a partial Y-chromosome genomic library and showing homology with the X and Y chromosomes and with an undetermined number of autosomes, detected two Y-specific restriction fragment length variants on male DNA that had been digested with Taq I and Eco RI. These variants may have been generated through a deletion-insertion mechanism and their pattern of holoandric transmission indicates that they represent a two-allele Y-linked polymorphism (RFLP). By means of DNA from patients with inborn deletions in chromosome Y, this polymorphic DNA site was mapped to the interval Yq11.1-Yq11.22. The frequency of the rarest allele was about 35 percent in Algerian and Sardinian human males, whereas it was only 4 percent among Northern Europeans. The p12f2 probe also detected Y-specific DNA fragments in the gorilla and chimpanzee. In view of the monosomy of the Y chromosome in mammalian species, Y-linked RFLP's may prove to be more useful than autosomal or X-linked markers in estimating genetic distances within and between species.

[Juvenile granulosa-cell tumor: clinical and molecular expression]. / Tumeurs juvéniles de la granulosa: expression clinique et moléculaire.

Ovarian sex cord-stromal tumors are rare tumors that originate from the nongerminal cells of ovary. Two decades ago, the identification of juvenile granulosa-cell tumors (GCT), as a specific entity inside this group, allowed a better treatment of these tumors in children. However, little data have been reported on the natural course of the disease and reliable prognostic factors have not been yet defined. We here review the clinical and genetics aspects of granulosa tumors, based on a series of 40 children. This national collaborative study involved the French Society of Children Cancer and eight clinical departments of pediatric endocrinology. We found that early diagnosis of a tumor, revealed by clinical signs of hyperoestrogeny, is an important prognostic factor. The pathophysiology of these tumors is still debatable and several cellular- and molecular-abnormal signals could be implicated in their development. The role of growth factors and oncogenes through the signaling pathway of MAP kinase is still discussed. According to our data, FSH signaling-transduction pathway, such as a constitutionally activated Galphas, could also be implicated in the induction of granulosa cell proliferation and seems to modulate the invasiveness of the tumor. Last, we have described a low-expression pattern or an extinction of an ovarian-determination gene, FOXL2, which is related to a worse prognosis of this tumor.

The mutations and potential targets of the forkhead transcription factor FOXL2.

Mutations of FOXL2, a gene encoding a forkhead transcription factor, have been shown to cause the blepharophimosis-ptosis-epicanthus inversus syndrome (BPES). This genetic disorder is characterized by eyelid and mild craniofacial abnormalities that can appear associated with premature ovarian failure. FOXL2 is one of the earliest ovarian markers and it offers, along with its targets, an excellent model to study ovarian development and function in normal and pathological conditions. In this review we summarize recent data concerning FOXL2, its mutations and its potential targets. Indeed, many mutations have been described in the coding sequence of FOXL2. Among them, polyalanine expansions and premature nonsense mutations have been shown to induce protein aggregation. In the context of the ovary, FOXL2 has been suggested to be involved in the regulation of cholesterol and steroid metabolism, apoptosis, reactive oxygen species detoxification and inflammation processes. The elucidation of the impact of FOXL2 mutations on its function will allow a better understanding of the pathogenic mechanisms underlying the BPES phenotype.

[Genetic analysis of premature ovarian failure: role of forkhead and TGF-beta genes]. / Analyse génétique des insuffisances ovariennes prématurées: implication des gènes forkhead et TGF-beta.

Premature ovarian failure is a common pathology affecting 1% of women. Although multiple etiologies have been described the majority of cases are idiopathic. Forkhead transcription factors as FOXL2 and FOXO3A are of particular interest in the research of genetic factors related with the pathology as they are present in diverse developmental pathways and ovarian physiology. Similarly, some TGF-beta factors (i.e. BMP 15 and GDF-9) have been demonstrated to play a key role in the regulation, at ovarian level, of female reproduction. In recent years numerous studies have been performed in order to elucidate the implication of these factors in the ovarian physiopathology. The aim of this manuscript is to describe some of these advances in the context of premature ovarian failure.

GermOnline, a cross-species community knowledgebase on germ cell differentiation.

GermOnline provides information and microarray expression data for genes involved in mitosis and meiosis, gamete formation and germ line development across species. The database has been developed, and is being curated and updated, by life scientists in cooperation with bioinformaticists. Information is contributed through an online form using free text, images and the controlled vocabulary developed by the GeneOntology Consortium. Authors provide up to three references in support of their contribution. The database is governed by an international board of scientists to ensure a standardized data format and the highest quality of GermOnline's information content. Release 2.0 provides exclusive access to microarray expression data from Saccharomyces cerevisiae and Rattus norvegicus, as well as curated information on approximately 700 genes from various organisms. The locus report pages include links to external databases that contain relevant annotation, microarray expression and proteome data. Conversely, the Saccharomyces Genome Database (SGD), S.cerevisiae GeneDB and Swiss-Prot link to the budding yeast section of GermOnline from their respective locus pages. GermOnline, a fully operational prototype subject-oriented knowledgebase designed for community annotation and array data visualization, is accessible at http://www.germonline.org. The target audience includes researchers who work on mitotic cell division, meiosis, gametogenesis, germ line development, human reproductive health and comparative genomics.

Cytogenetic study of a European Burkitt's lymphoma cell line.

The chromosomes of an Epstein-Barr virus-negative European Burkitt's lymphoma cell line were studied. All the cells carried the t(8;14) translocation. One clone had 51 chromosomes and was (+1,+7,+16,+15,+21), whereas another clone also had 51 chromosomes but was (+1q+,+7,+16,+15,+21). A third clone had 46 chromosomes (4q+/-).

Extensive sequence homologies between Y and other human chromosomes.

Twenty-six human Y-chromosome-derived DNA sequences, free of repetitive material, were used to probe male and female genomic blots. We present data from a detailed analysis and chromosomal location of the bands detected by such probes, which demonstrate extensive DNA sequence homology between the mammalian sex chromosomes and autosomes. Under stringent conditions, nine Y-derived probes reacted exclusively with the Y chromosome, 12 probes detected homologous sequences present on both the Y and the X, four probes detected homologies between Y and autosome(s) without any X counterpart and, finally, one probe hybridized to homologous sequences on Y, X and autosome(s). These data are consistent with the hypothesis of a common evolutionary origin for the mammalian sex chromosomes and reveal structural similarities between Y-located and autosomal non-repetitive sequences.

Formal genetics of the HL-A region.

The extreme polymorphism of the HL-A system is due to the presence of two (SD1, SD2) and perhaps three linked polyallelic genes. The distinction of "bridging antibodies" (reacting with several HL-A specificities recognizing separate sites on the HL-A molecule) from the main HL-A determinant as it is demonstrated by absorption/inhibition experiments increases this complexity. The HL-A linkage group is composed of other systems: LD1, LD2, PGM3, ADA (?), P, ME1, IPO-B and possibly a "hay fever gene". No gametic or zygotic selection was found in spite of the presence of HL-A antigens on spermatozoa. Mixed lymphocyte reaction (MLR) is principally governed by LD genes. The main (LD2) gene is probably situated outside the interval SD1, SD2, near SD2. Other LD genes (LD1 inside the interval SD1-SD2 and LD3) are suspected. The presence of an immune response gene (Ir) has not yet been demonstrated although several diseases associated with specific SD2 antigens are known. These different genes (SD1, SD2, LD1, LD2, LD3 and Ir) probably form a functional unit in the allo-immunozation.

NSPc1, a novel mammalian Polycomb gene, is expressed in neural crest-derived structures of the peripheral nervous system.

Anterior-posterior (A-P) patterning is a key element in early embryonic development. Polycomb group (PcG) genes act as transcriptional repressors to regulate A-P patterning by either directly or indirectly controlling the coordinated expression of the HOM/Hox homeobox (Curr. Opin. Genet. Dev. 7 (1997) 488; Trends Genet. 13 (1997) 167). We describe the isolation and characterization of a novel mammalian PcG gene, termed Nervous System Polycomb-1 (NSPc1). Human and mouse NSPc1 genes encode proteins with an N-terminal RING finger domain and share homology with Drosophila melanogaster lethal(3)73Ah and the mammalian Mel18 and Bmi1 genes. Transcripts are observed at 10 dpc in the otic vesicle, urogenital bud and dorsal root ganglia. At 11.5 dpc, transcripts are present in a subset of neural crest cell derivatives of the peripheral nervous system, and in the neural tube. NSPc1 expression is ubiquitous in adult tissue.