Antonino Forabosco

Mutations in GPC3, a glypican gene, cause the Simpson-Golabi-Behmel overgrowth syndrome

Simpson-Golabi-Behmel syndrome (SGBS) is an X-linked condition characterized by pre-and postnatal overgrowth with visceral and skeletal anomalies. To identify the causative gene, breakpoints in two female patients with X;autosome translocations were identified. The breakpoints occur near the 5′, and 3′, ends of a gene, GPC3, that spans more than 500 kilobases in Xq26; in three families, different microdeletions encompassing exons cosegregate with SGBS. GPC3 encodes a putative extracellular proteoglycan, glypican 3, that is inferred to play an important role in growth control in embryonic mesodermal tissues in which it is selectively expressed. Initial western- and ligand-blotting experiments suggest that glypican 3 forms a complex with insulin-like growth factor 2 (IGF2), and might thereby modulate IGF2 action.

Gpc3 expression correlates with the phenotype of the Simpson‐Golabi‐Behmel syndrome

Interest in glypican‐3 (GPC3), a member of the glypican‐related integral membrane heparan sulfate proteoglycans (GRIPS) family, has increased with the finding that it is mutated in the Simpson‐Golabi‐Behmel overgrowth syndrome (Pilia et al. [1996] Nat. Genet. 12:241–247). The working model suggested that the membrane‐bound protein acts locally to limit tissue and organ growth and that it may function by interacting with insulin‐like growth factor 2 (IGF2) to limit its local effective level. Here we have tested two predictions of the model. In situ hybridization with the mouse gene cDNA was used to study the expression pattern during embryonic and fetal development. In agreement with predictions, the gene is expressed in precisely the organs that overgrow in its absence; and the patterns of expression of Gpc3 and those reported for Igf2 are strictly correlated. Dev. Dyn. 1998; 213:431–439.

Foxl2 functions in sex determination and histogenesis throughout mouse ovary development

BackgroundPartial loss of function of the transcription factor FOXL2 leads to premature ovarian failure in women. In animal models, Foxl2 is required for maintenance, and possibly induction, of female sex determination independently of other critical genes, e.g., Rspo1. Here we report expression profiling of mouse ovaries that lack Foxl2 alone or in combination with Wnt4 or Kit/c-Kit.ResultsFollowing Foxl2 loss, early testis genes (including Inhbb, Dhh, and Sox9) and several novel ovarian genes were consistently dysregulated during embryonic development. In the absence of Foxl2, expression changes affecting a large fraction of pathways were opposite those observed in Wnt4-null ovaries, reinforcing the notion that these genes have complementary actions in ovary development. Loss of one copy of Foxl2 revealed strong gene dosage sensitivity, with molecular anomalies that were milder but resembled ovaries lacking both Foxl2 alleles. Furthermore, a Foxl2 transgene disrupted embryonic testis differentiation and increased the levels of key female markers.ConclusionThe results, including a comprehensive principal component analysis, 1) support the proposal of dose-dependent Foxl2 function and anti-testis action throughout ovary differentiation; and 2) identify candidate genes for roles in sex determination independent of FOXL2 (e.g., the transcription factors IRX3 and ZBTB7C) and in the generation of the ovarian reserve downstream of FOXL2 (e.g., the cadherin-domain protein CLSTN2 and the sphingomyelin synthase SGMS2). The gene inventory is a first step toward the identification of the full range of pathways with partly autonomous roles in ovary development, and thus provides a framework to analyze the genetic bases of female fertility.

Gonadoblastoma in Turner syndrome and Y-chromosome-derived material.

The identification of Y‐chromosome material is important in females with Ullrich–Turner syndrome (UTS) due to the risk of developing gonadoblastoma or other gonadal tumors. There is controversy regarding the frequency of the Y‐chromosome‐derived material and the occurrence of gonadoblastoma in these patients. The aim of our study was to evaluate a large number of patients with UTS, followed before and during the pubertal age for the prevalence of Y‐chromosome derived material, the occurrence of gonadoblastoma, and the incidence of possible neoplastic degeneration. An unselected series of 171 patients with UTS (1–34 years old), diagnosed cytogenetically, was studied for Y‐chromosome markers (SRY and Y‐centromeric DYZ3 repeats). The follow‐up was of 2–22 years; 101 of these patients were followed during pubertal age. Y‐chromosome material was found in 14 patients (8%): 12 of these were gonadectomized (2.8–25.9 years). A gonadoblastoma was detected in four patients under 16 years of age: in two, Y‐material was detected only at molecular analysis (at conventional cytogenetic analysis, one was included in the 45,X group and one in the X + mar group) and one had also an immature teratoma and an endodermal sinus carcinoma. The prevalence of gonadoblastoma in our series of gonadectomized UTS patients with Y‐positive material was of 33.3% (4/12). Our data suggest that the age of appearance and the possibility of malignant degeneration of gonadoblastoma can occur early in life. These patients, in particular those with 45,X or a marker chromosome may benefit from molecular screening to detect the presence of Y‐chromosome material; PCR is a rapid and inexpensive technique. At the moment, laparoscopy and preventive gonadectomy performed as soon as possible remain the procedures of choice for patients with UTS, when Y‐chromosome has been identified, as we are still unable to predict a future malignant evolution of gonadoblastoma.

Aging of oocyte, ovary, and human reproduction

Abstract: We review age‐related changes in the ovary and their effect on female fertility, with particular emphasis on follicle formation, follicle dynamics, and oocyte quality. The evidence indicates that the developmental processes leading to follicle formation set the rules determining follicle quiescence and growth. This regulatory system is maintained until menopause and is directly affected in at least some models of premature ovarian failure (POF), most strikingly in the Foxl2 mouse knockout, a model of human POF with monogenic etiology (blepharophimosis/ptosis/epicanthus inversus syndrome). Several lines of evidence indicate that if the ovarian germ cell lineage maintains regenerative potential, as recently suggested in the mouse, a role in follicle dynamics for germ stem cells, if any, is likely indirect or secondary. In addition, age‐related variations in oocyte quality in animal models suggest that reproductive competence is acquired progressively and might depend on parallel growth and differentiation of follicle cells and stroma. Genomewide analyses of the mouse oocyte transcriptome have begun to be used to systematically investigate the mechanisms of reproductive competence that are altered with aging. Investigative and therapeutic strategies can benefit from considering the role of continuous interactions between follicle cells and oocytes from the beginning of histogenesis to full maturation.

A panel of subchromosomal painting libraries representing over 300 regions of the human genome.

DNA samples from about 100 human-hamster somatic cell hybrids, previously characterized by conventional banding techniques, were amplified with dual-Alu PCR. The products were then used as probes in FISH experiments on normal human metaphases for an accurate cytogenetic characterization of the human material retained in each hybrid. In addition to entire chromosomes, most hybrids were found to contain one or a few chromosome fragments, as a result of rearrangements that had occurred in vitro. Forty additional primary hybrids, in which conventional cytogenetic analysis failed to reveal any complete human chromosome, contained many human chromosome fragments. More than 300 chromosome fragments were scored and their precise chromosomal location recorded. We show data indicating that subchromosomal painting libraries generated from these hybrids can be favorably used in the fine characterization of chromosomal rearrangements encountered in clinical cytogenetics or in tumor cytogenetics, and in tracking chromosomal changes that occurred in primate evolution.

Incidence of non-age-dependent chromosomal abnormalities: a population-based study on 88965 amniocenteses

Current knowledge about the incidence of chromosomal abnormalities in the general population comes from studies in newborns carried out in the 70s, before the era of widespread prenatal diagnosis. In the following years, data on frequency of chromosomal abnormalities in the second trimester of pregnancy have been used in conjunction with the data on the natural history of chromosomally abnormal fetuses to infer maternal age-specific rates of cytogenetic abnormalities in live-born infants. Starting from the data gathered in 1995–1996 from all Italian cytogenetic laboratories (with 92% compliance to the study), we have compared the frequency of chromosomal abnormalities at amniocentesis in cases with maternal age of ≥35 years (51 758 individuals) and cases with maternal age of <35 years (37 207 cases). The comparison confirmed the age-dependency of aneuploidies, whereas none of the structural abnormalities showed age-related differences. Furthermore, among the mosaic aneuploidies, trisomy 21 and 45,X/46,XX were found with a significantly higher incidence in older women. Chromosomal abnormalities that showed no significant difference between the two groups were summed for the overall national cohort, providing a general estimate of the incidence in the second trimester of pregnancy. The data provide critical background information for prenatal genetic counseling and for the planning of health care policy.

Noonan-like syndrome with loose anagen hair: A new syndrome?

We present three children with short stature, the same facial phenotype, macrocephaly, enlarged cerebral spinal fluid spaces, short neck with redundant skin, severe GH deficiency, mild psychomotor delay with attention deficit/hyperactivity disorder (ADHD), mild dilatation of the pulmonary root in two of them, and a unique combination of ectodermal abnormalities. Their appearance, not completely typical of Noonan syndrome, the behavioral phenotype, GH deficiency, darkly pigmented and hairless skin, and the unusual aspect of the hair, defined as loose anagen hair syndrome did not fit any known condition. We postulate that these children may represent a distinct, previously unreported syndrome that we would name “Noonan‐like syndrome with loose anagen hair”.

Emx2 developmental expression in the primordia of the reproductive and excretory systems.

Abstract The development of the urogenital system has always attracted many investigators owing to the peculiar aspects of the embryology of the reproductive and excretory organs and to the high number of congenital anomalies related to these structures. It is remarkable because of the common origin of the kidneys, gonads, and genital tracts from the intermediate mesoderm and because differentiation of these organs involves extensive mesenchyme to epithelium transition. Our knowledge about the molecular mechanisms controlling the differentiation of these diverse structures from the same precursor has taken advantage of gene expression data and gene-targeting experiments using genes with a specific expression pattern in the urogenital system. A more detailed function in kidney development has been postulated for transcription factors such as WT-1, Pax-2 or other molecules such as glial cell line-derived neurotrophic factor (GDNF), Wnt-4, c-ret. In the present work we have described the expression pattern of the homeobox-containing gene Emx2 during the development of the urogenital system in mouse embryos. We have found that Emx2 is expressed in the early primordia of the organs that will form the excretory and reproductive systems. In particular we have found that Emx2 is expressed in the epithelial components of pronephros and mesonephros, in Wolffian and Müllerian ducts, in the ureteric buds with their branches and in the early epithelial structures derived from metanephrogenic mesenchyme. Emx2 is also intensely expressed in the ”bipotential” or ”indifferent” gonads and ovaries. These data and the recent finding that Emx2 homozygous mutant mice die soon after birth because of the absence of kidneys indicate an essential role of Emx2 in the morphogenesis of the urogenital system.

Determination and Stability of Gonadal Sex

The discovery that the SRY gene induces male sex in humans and other mammals led to speculation about a possible equivalent for female sex. But females are proving to be more complicated. Several master genes appear to be autonomously involved, and female sex determination seems to remain relatively labile. Partial loss of function of the transcription factor FOXL2 leads to premature ovarian failure in women; and in animal models, Foxl2 is required for folliculogenesis as well as for maintenance, and possibly induction, of female sex determination. In the germ line, oocytes apparently form normally even in the absence of Foxl2, dependent on genes that include female-specific factors such as Fig-alpha, Nobox, etc. In the soma, ablation of Foxl2 or the independently expressed gene Wnt4 (likely downstream of Rspo1) can produce partial testis differentiation in XX mice, and the double knockout results in the formation of tubules and spermatogonia. This indicates that at least 2 autonomous ovarian pathways are required to antagonize testis differentiation in females, a finding that is being increasingly corroborated by studies in goats and nonmammalian vertebrates. In recent expression profiling of mouse ovaries that lack Foxl2 alone or in combination with Wnt4 or Kit/c-Kit, we found that following Foxl2 loss, early testis genes (including the downstream effector of Sry, Sox9) and several novel ovarian genes were consistently dysregulated during embryo-fetal development. The results support the proposal of dose-dependent Foxl2 function and antitestis action. A partial working model for somatic development and sex determination is presented in which Sox9 is a direct antagonist of Foxl2 in the supporting cell lineage.