GREB1L variants in familial and sporadic hereditary urogenital adysplasia and Mayer-Rokitansky-Kuster-Hauser syndrome.

Congenital uterine anomalies (CUA) may have major impacts on the health and social well-being of affected individuals. Their expressivity is variable, with the most severe end of the spectrum being the absence of any fully or unilaterally developed uterus (aplastic uterus), which is a major feature in Mayer-Rokitansky-Kuster-Hauser syndrome (MRKH). So far, etiologies of CUA remain largely unknown. As reports of familial occurrences argue for strong genetic contributors in some cases, we performed whole exome sequencing in nine multiplex families with recurrence of uterine and kidney malformations, a condition called hereditary urogenital adysplasia. Heterozygous likely causative variants in the gene GREB1L were identified in four of these families, confirming GREB1L as an important gene for proper uterine and kidney development. The apparent mode of inheritance was autosomal dominant with incomplete penetrance. The four families included fetuses with uterovaginal aplasia and bilateral renal agenesis, highlighting the importance to investigate GREB1L in such phenotypes. Subsequent sequencing of the gene in a cohort of 68 individuals with MRKH syndrome or uterine malformation (mostly sporadic cases) identified six additional variants of unknown significance. We therefore conclude that heterozygous GREB1L variants contribute to MRKH syndrome and this probably requires additional genetic or environmental factors for full penetrance.

Involvement of ITIH5, a candidate gene for congenital uterovaginal aplasia (Mayer-Rokitansky-Küster-Hauser syndrome), in female genital tract development.

The ITI (inter-trypsine inhibitor) gene family includes five genes (ITIH1 to ITIH5) that encode proteins involved in the dynamics of the extracellular matrix (ECM). ITIH5 was found inactivated by partial deletion in a case of congenital uterovaginal aplasia, a human rare disease also called Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome. The aim of the present study was to analyze the expression of ITIH5 in the uterus in adult life and during embryogenesis in order to establish the involvement of this gene in both normal and pathological conditions of uterus development. This was achieved in mice by reverse transcription-quantitative PCR, whole-mount hybridization, and Western blot analysis. Itih5 expression was much stronger in female genital tract primordia (Müllerian ducts) and derivatives than elsewhere in the body. This gene was strongly expressed during pregnancy and development of the female genital tract, indicating that the encoded protein probably had an important function in the uterus during these periods. Two different specific isoforms of the protein were detected in Müllerian derivatives during embryogenesis and in adults. Although ITIH genes are expected to be predominantly expressed in the liver, ITIH5 is mainly expressed in the uterus during development and adult life. This tends to indicate an additional and specific role of this gene in the female reproductive tract, and furthermore reinforces ITIH5 as a putative candidate gene for MRKH syndrome.

ZFPIP/Zfp462 is involved in P19 cell pluripotency and in their neuronal fate.

The nuclear zinc finger protein ZFPIP/Zfp462 is an important factor involved in cell division during the early embryonic development of vertebrates. In pluripotent P19 cells, ZFPIP/Zfp462 takes part in cell proliferation, likely via its role in maintaining chromatin structure. To further define the function of ZFPIP/Zfp462 in the mechanisms of pluripotency and cell differentiation, we constructed a stable P19 cell line in which ZFPIP/Zfp462 knockdown is inducible. We report that ZFPIP/Zfp462 was vital for mitosis and self-renewal in pluripotent P19 cells. Its depletion induced substantial decreases in the expression of the pluripotency genes Nanog, Oct4 and Sox2 and was associated with the transient expression of specific neuronal differentiation markers. We also demonstrated that ZFPIP/Zfp462 expression appears to be unnecessary after neuronal differentiation is induced in P19 cells. Taken together, our results strongly suggest that ZFPIP/Zfp462 is a key chromatin factor involved in maintaining P19 pluripotency and in the early mechanisms of neural differentiation but that it is dispensable in differentiated P19 cells.

Utero-vaginal aplasia (Mayer-Rokitansky-Küster-Hauser syndrome) associated with deletions in known DiGeorge or DiGeorge-like loci.

BACKGROUND: Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is characterized by congenital aplasia of the uterus and the upper part of the vagina in women showing normal development of secondary sexual characteristics and a normal 46, XX karyotype. The uterovaginal aplasia is either isolated (type I) or more frequently associated with other malformations (type II or Müllerian Renal Cervico-thoracic Somite (MURCS) association), some of which belong to the malformation spectrum of DiGeorge phenotype (DGS). Its etiology remains poorly understood. Thus the phenotypic manifestations of MRKH and DGS overlap suggesting a possible genetic link. This would potentially have clinical consequences. METHODS: We searched DiGeorge critical chromosomal regions for chromosomal anomalies in a cohort of 57 subjects with uterovaginal aplasia (55 women and 2 aborted fetuses). For this candidate locus approach, we used a multiplex ligation-dependent probe amplification (MLPA) assay based on a kit designed for investigation of the chromosomal regions known to be involved in DGS.The deletions detected were validated by Duplex PCR/liquid chromatography (DP/LC) and/or array-CGH analysis. RESULTS: We found deletions in four probands within the four chromosomal loci 4q34-qter, 8p23.1, 10p14 and 22q11.2 implicated in almost all cases of DGS syndrome. CONCLUSION: Uterovaginal aplasia appears to be an additional feature of the broad spectrum of the DGS phenotype. The DiGeorge critical chromosomal regions may be candidate loci for a subset of MRKH syndrome (MURCS association) individuals. However, the genes mapping at the sites of these deletions involved in uterovaginal anomalies remain to be determined. These findings have consequences for clinical investigations, the care of patients and their relatives, and genetic counseling.

Involvement of ZFPIP/Zfp462 in chromatin integrity and survival of P19 pluripotent cells.

Toti- or pluripotent cells proliferation and/or differentiation have been shown to be strongly related to nuclear chromatin organization and structure over the last past years. We have recently identified ZFPIP/Zfp462 as a zinc finger nuclear factor necessary for correct cell division during early embryonic developmental steps of vertebrates. We thus questioned whether this factor was playing a general role during cell division or if it was somehow involved in embryonic cell fate or differentiation. To achieve this goal, we performed a knock-down experiment in the pluripotent P19 and differentiated 3T3 cell lines, both expressing endogenous ZFPIP/Zfp462. Using specific shRNA directed against ZFPIP/Zfp462 transcripts, we demonstrated that depletion of this protein induced cell death in P19 but had no effect in 3T3 cells. In addition, in the absence of the protein, the P19 cells exhibited a complete destructuration of pericentromeric domains associated with a redistribution of the HP1alpha proteins and an increase in DNA satellites transcribed RNAs level. These data suggested an instrumental role of ZFPIP/Zfp462 in maintaining the chromatin structure of pluripotent cells.

The developing female genital tract: from genetics to epigenetics.

The mammalian female reproductive tract develops from the Mullerian ducts which differentiate, in a cranial to caudal direction, into oviducts, uterine horns, cervix and the anterior vagina. The developmental processes taking place during this organogenesis are notably under the control of steroid hormones, such as members of the Wnt and Hox families, which regulate key developmental genes. At later stages, steroid hormones also participate in the development of the female genital tract. Chemical compounds homologous to steroids can thus act as agonists or antagonists in fetuses exposed to them. These so-called endocrine disruptors are nowadays found in increasing amounts in the environment and may therefore have a particular impact on such developing organs. Epidemiological studies have revealed that endocrine disruptors have had drastic effects on female health and fertility during the last decades. Furthermore, these adverse effects might be transmitted to subsequent generations through epigenetic modifications. Given the potential hazard of inherited epigenetic marks altering reproduction and/or human health, such molecular mechanisms must be urgently investigated. This review aims to summarize the cellular and molecular mechanisms involved in female genital tract development, to highlight key genes involved in this process and to present epigenetic mechanisms triggered by endocrine disruptors and their consequences in regard to female reproductive tract development.

Inducible expression of beta defensins by human respiratory epithelial cells exposed to Aspergillus fumigatus organisms.

BACKGROUND: Aspergillus fumigatus, a saprophytic mould, is responsible for life-threatening, invasive pulmonary diseases in immunocompromised hosts. The role of the airway epithelium involves a complex interaction with the inhaled pathogen. Antimicrobial peptides with direct antifungal and chemotactic activities may boost antifungal immune response. RESULTS: The inducible expression of defensins by human bronchial epithelial 16HBE cells and A549 pneumocyte cells exposed to A. fumigatus was investigated. Using RT-PCR and real time PCR, we showed an activation of hBD2 and hBD9 defensin genes: the expression was higher in cells exposed to swollen conidia (SC), compared to resting conidia (RC) or hyphal fragments (HF). The kinetics of defensin expression was different for each one, evoking a putative distinct function for each investigated defensin. The decrease of defensin expression in the presence of heat-inactivated serum indicated a possible link between defensins and the proteins of the host complement system. The presence of defensin peptide hBD2 was revealed using immunofluorescence that showed a punctual cytoplasmic and perinuclear staining. Quantification of the cells stained with anti hBD2 antibody demonstrated that SC induced a greater number of cells that synthesized hBD2, compared to RC or HF. Labelling of the cells with anti-hBD-2 antibody showed a positive immunofluorescence signal around RC or SC in contrast to HF. This suggests co-localisation of hBD2 and digested conidia. The HBD2 level was highest in the supernatants of cells exposed to SC, as was determined by sandwich ELISA. Experiments using neutralising anti-interleukine-1beta antibody reflect the autocrine mechanism of defensin expression induced by SC. Investigation of defensin expression at transcriptional and post-transcriptional levels demonstrated the requirement of transcription as well as new protein synthesis during A. fumigatus defensin induction. Finally, induced defensin expression in primary culture of human respiratory cells exposed to A. fumigatus points to the biological significance of described phenomena. CONCLUSION: Our findings provide evidence that respiratory epithelium might play an important role in the immune response during Aspergillus infection. Understanding the mechanisms of regulation of defensin expression may thus lead to new approaches that could enhance expression of antimicrobial peptides for potential therapeutic use during aspergillosis treatment.

Identification of a new type of PBX1 partner that contains zinc finger motifs and inhibits the binding of HOXA9-PBX1 to DNA.

PBX1 belongs to the TALE-class of homeodomain protein and has a wide functional diversity during development. Indeed, PBX1 is required for haematopoiesis as well as for multiple developmental processes such as skeletal patterning and organogenesis. It has furthermore been shown that PBX1 functions as a HOX cofactor during development. More recent data suggest that PBX1 may act even more broadly by modulating the activity of non-homeodomain transcription factors. To better understand molecular mechanisms triggered by PBX1 during female genital tract development, we searched for additional PBX1 partners that might be involved in this process. Using a two hybrid screen, we identified a new PBX1 interacting protein containing several zinc finger motifs that we called ZFPIP for Zinc Finger PBX1 Interacting Protein. We demonstrated that ZFPIP is expressed in embryonic female genital tract but also in other PBX1 expression domains such as the developing head and the limb buds. We further showed that ZFPIP is able to bind physically and in vivo to PBX1 and moreover, that it prevents the binding of HOXA9/PBX complexes to their consensus DNA site. We suggest that ZFPIP is a new type of PBX1 partner that could participate in PBX1 function during several developmental pathways.

Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome.

The Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is characterized by congenital aplasia of the uterus and the upper part (2/3) of the vagina in women showing normal development of secondary sexual characteristics and a normal 46, XX karyotype. It affects at least 1 out of 4500 women. MRKH may be isolated (type I) but it is more frequently associated with renal, vertebral, and, to a lesser extent, auditory and cardiac defects (MRKH type II or MURCS association). The first sign of MRKH syndrome is a primary amenorrhea in young women presenting otherwise with normal development of secondary sexual characteristics and normal external genitalia, with normal and functional ovaries, and karyotype 46, XX without visible chromosomal anomaly. The phenotypic manifestations of MRKH syndrome overlap with various other syndromes or associations and thus require accurate delineation. For a long time the syndrome has been considered as a sporadic anomaly, but increasing number of familial cases now support the hypothesis of a genetic cause. In familial cases, the syndrome appears to be transmitted as an autosomal dominant trait with incomplete penetrance and variable expressivity. This suggests the involvement of either mutations in a major developmental gene or a limited chromosomal imbalance. However, the etiology of MRKH syndrome still remains unclear. Treatment of vaginal aplasia, which consists in creation of a neovagina, can be offered to allow sexual intercourse. As psychological distress is very important in young women with MRKH, it is essential for the patients and their families to attend counseling before and throughout treatment.

Phenotypic variability of a 4q34-->qter inherited deletion: MRKH syndrome in the daughter, cardiac defect and Fallopian tube cancer in the mother.

Terminal deletions of the long arm of chromosome 4 are associated with a recognizable phenotype consisting of dysmorphic facial features, cleft palate, upper and lower limb malformations, cardiac defects and growth and mental retardation. Here we report on two female patients, a mother and her daughter, carrying the same 4q34-->qter deletion but presenting with a different phenotype. The mother's presentation is consistent with previous findings in patients with terminal deletions of the long arm of chromosome 4. However, she presented at the age of 54years with bilateral serous carcinoma of the Fallopian tubes, a rare gynaecologic cancer that might be attributed to the haploinsufficiency of the tumor suppressor gene FAT. The daughter presented isolated congenital aplasia of the uterus and vagina, the prime feature of the MRKH syndrome. This has not been described before in association with a 46,XX,del(4)(q34qter).

Role of HOXA7 to HOXA13 and PBX1 genes in various forms of MRKH syndrome (congenital absence of uterus and vagina).

The Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome refers to the congenital absence or severe hypoplasia of the female genital tract, often described as uterovaginal aplasia which is the prime feature of the syndrome. It is the second cause of primary amenorrhea after gonadal dysgenesis and occurs in approximately 1 in 4500 women. Aetiology of this syndrome remains poorly understood. Frequent association of other malformations with the MRKH syndrome, involving kidneys, skeleton and ears, suggests the involvement of major developmental genes such as those of the HOX family. Indeed mammalian HOX genes are well known for their crucial role during embryogenesis, particularly in axial skeleton, hindbrain and limb development. More recently, their involvement in organogenesis has been demonstrated notably during urogenital differentiation. Although null mutations of HOX genes in animal models do not lead to MRKH-like phenotypes, dominant mutations in their coding sequences or aberrant expression due to mutated regulatory regions could well account for it. Sequence analysis of coding regions of HOX candidate genes and of PBX1, a likely HOX cofactor during Müllerian duct differentiation and kidney morphogenesis, did not reveal any mutation in patients showing various forms of MRKH syndrome. This tends to show that HOX genes are not involved in MRKH syndrome. However it does not exclude that other mechanisms leading to HOX dysfunction may account for the syndrome.

The Mayer-Rokitansky-Küster-Hauser syndrome (congenital absence of uterus and vagina)--phenotypic manifestations and genetic approaches.

The Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome affects at least 1 out of 4500 women and has for a long time been considered as a sporadic anomaly. Congenital absence of upper vagina and uterus is the prime feature of the disease which, in addition, is often found associated with unilateral renal agenesis or adysplasia as well as skeletal malformations (MURCS association). The phenotypic manifestations of MRKH overlap various other syndromes or associations and thus require accurate delineation. Since MRKH manifests itself in males, the term GRES syndrome (Genital, Renal, Ear, Skeletal) might be more appropriate when applied to both sexes. The MRKH syndrome, when described in familial aggregates, seems to be transmitted as an autosomal dominant trait with an incomplete degree of penetrance and variable expressivity. This suggests the involvement of either mutations in a major developmental gene or a limited chromosomal deletion. Until recently progress in understanding the genetics of MRKH syndrome has been slow, however, now HOX genes have been shown to play key roles in body patterning and organogenesis, and in particular during genital tract development. Expression and/or function defects of one or several HOX genes may account for this syndrome.

Candidate Sertoli cell specific promoter element for a TGFbeta family member (Amh) and a 3' UTR enhancer/repressor for the same gene.

Comparison between 400 nt of mouse DNA sequence immediately upstream (5') of the coding sequence of the Sertoli cell expressed genes, Amh and Tsx, identified a 33 nt sequence with a significant identity: this was considered to be a candidate Sertoli specific regulatory element (SSRE). Another highly conserved sequence has been identified immediately downstream (3') of the Amh polyadenylation signal (DSRE). The action and specificity of these two putative regulatory elements, inserted into a reporter vector (pd2EGFP), has been investigated by transient transfection of mouse fibroblast and Sertoli cells. When combined together or alone, with a minimal thymidine kinase (Tk) promoter, SSRE showed a weak incremental action on the expression of the reporter gene, regardless of the cell type: in contrast there was a 2- to 3-fold decrease when DSRE was present. However, in the absence of Tk there was evidence for a strong synergy between SSRE and DSRE, which was significantly greater in the Sertoli as compared with the fibroblast cells. These results support the view that SSRE exhibits a degree of Sertoli specificity and acts synergistically with DSRE in controlling the expression of Amh.

PBX1 intracellular localization is independent of MEIS1 in epithelial cells of the developing female genital tract.

While studies have highlighted the role of HOXA9-13 and PBX1 homeobox genes during the development of the female genital tract, the molecular mechanisms triggered by these genes are incompletely elucidated. In several developmental pathways, PBX1 binds to MEINOX family members in the cytoplasm to be imported into the nucleus where they associate with HOX proteins to form a higher complex that modulates gene expression. This concept has been challenged by a recent report showing that in some cell cultures, PBX1 nuclear localization might be regulated independently of MEINOX proteins (Kilstrup-Nielsen et al., 2003). Our work gives the first illustration of this alternative mechanism in an organogenesis process. Indeed, we show that PBX1 is mostly cytoplasmic in epithelial endometrial cells of the developing female genital tract despite the nuclear localization of MEIS1. We thus provide evidence for a control of PBX1 intracellular distribution which is independent of MEINOX proteins, but is cell cycle correlated.

In vivo magnetic resonance imaging of large spontaneous aortic aneurysms in old apolipoprotein E-deficient mice.

UNLABELLED: Old ApoE-deficient mice were studied in vivo by magnetic resonance imaging (MRI) to prospectively evaluate vascular remodeling associated with atherosclerotic lesions. MATERIAL AND METHODS: Old female ApoE-/- mice on a normal diet were followed by MRI at 2 Tesla for a 3-month period and killed for histopathology. Aortic dimensions were measured and compared. RESULTS: High-quality in vivo MR images were obtained at 2 Tesla with in plane spatial resolution of 86 X 86 microm2. On MRI, aortic lumen enlargement (>1.5-fold dilation) was seen in 10 of 13 mice, located predominantly in the suprarenal portion of the aorta. The mean maximal diameter of the aneurysms and of the aorta above and below the aneurysm were, respectively, 1.12 +/- 0.32 mm and 0.53 +/- 0.08 mm by MRI and 1.3+/- 0.41 mm and 0.55 +/- 0.15 mm by histology. Matched histologic cross-sections of the aortic wall showed medial degradation with rupture of the internal elastic lamina at multiple sites, associated with fibrolipidic plaque containing cholesterol crystals. CONCLUSIONS: Aortic lumen enlargement was diagnosed in old ApoE-/- mice at sites with advanced atherosclerotic plaques. MRI has potential both as an in vivo imaging technique for screening mouse models for vascular wall pathology and to follow arterial remodeling associated with the disease progression.

A conserved non-homeodomain Hoxa9 isoform interacting with CBP is co-expressed with the 'typical' Hoxa9 protein during embryogenesis.

Various Hox genes are known to produce alternative transcripts encoding different isoforms whose physiological relevance during development is not yet understood. In this work, we analysed two different Hoxa9 mRNAs encoding a full-length protein (Hoxa9) or a protein lacking the homeodomain (Hoxa9T). First, we demonstrated that these transcripts are conserved from birds to mammals. We then showed that both transcripts are present throughout embryogenesis and that Hoxa9T transcript is particularly abundant in embryonic genital tract, kidney, forelimb and tail. We further found that both isoforms are able to interact with CBP, suggesting a competition between Hoxa9 and Hoxa9T with this protein.

High-resolution magnetic resonance imaging at 2 Tesla: potential for atherosclerotic lesions exploration in the apolipoprotein E knockout mouse.

INTRODUCTION: The aim of the present study was to evaluate the potential of high-resolution MRI at 2 Tesla (T) for direct noninvasive imaging of the aortic wall in a mouse model of atherosclerosis. MATERIAL AND METHODS: A specific mouse antenna was developed and sequence parameters were adjusted. T(1)- and T2-weighted images of abdominal aorta were obtained at 2 T with a spatial resolution of 86 x 86 x 800 microm3 in vivo. With a dedicated small coil, ex vivo MRI of the aorta was performed with a spatial resolution of 54 x 54 x 520 microm3. RESULTS: In vivo, the aortic wall was clearly defined on T(2)-weighted images in 15 of 16 mice: along the aorta the lumen circumference ranged from 1.07 to 3.61 mm and mean wall thickness from 0.11 to 0.67 mm. In vivo measurements of plaque distribution were confirmed by ex vivo MR imaging and by histology, with a good correlation with histology regarding lumen circumference (r = 0.94) and wall thickness (r = 0.97). CONCLUSION: Magnetic resonance imaging at 2 T to analyze in vivo atherosclerotic lesions in mice is possible with a spatial resolution of 86 x 86 x 800 microm3 and thus can be used for noninvasive follow-up in evaluation of new drugs.