CLINICAL GENOMICS
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Owen M. Rennert, MD, Head, Section on Developmental Genomics Wai-Yee Chan, PhD, Adjunct Investigator |
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It is increasingly obvious that biological processes in living organisms are tightly regulated by networks of genes. The traditional one gene-one pathway is not appropriate in the study of complicated biological processes such as development. Our research uses functional genomic approaches to study germ cell and sexual development in normal and pathological states in order to understand the intricate regulatory mechanism of cellular proliferation and differentiation. Similarly, many seemingly nonhereditary diseases have now been shown to have a genetic component. In line with this observation, we have identified a genetic risk factor for pseudotumor cerebri in the Turkish population, in which heritable factors had not been previously identified. Functional genomic studies of germ cell development in spermatogenesis Wu, Baxendale, Pang, Johnson, Leung, Ruszczyk, Rennert, Chan; in collaboration with Dym, Ravindranath, Stitely Spermatogenesis is a tightly regulated process characterized by spermatogonial stem cells undergoing mitotic expansion, meiosis, and postmeiotic differentiation. The distinct morphological and biological characteristics of germ cells observed at different stages of spermatogenesis allow preparation of these cells in relatively pure form. In addition, animal models permit the study of arrest and reinitiation of spermatogonial differentiation, making spermatogenesis a unique model for studying stem cells and the genetic factors that regulate cellular proliferation and differentiation in general. By sequencing the respective SAGE (Serial Analysis of Gene Expression) libraries, we determined the transcriptome of mouse type A spermatogonia (Spga), pachytene spermatocytes (Spcy), and round spermatids (Sptd) purified by the STAPUT technique. We analyzed 444,015 SAGE tags derived from one Spga, two Spcy, and one Sptd library and identified 34,619 different species of transcripts among these SAGE tags. Among the transcripts, 42.6 percent were derived from known genes, 42.1 percent were uncharacterized cDNAs, and 15.3 percent had not been previously described. Analysis of the spermatocyte transcriptome indicates that the germ cell transcriptome could consist of more than 30,000 transcripts, different from what has been observed for other cells (Zhang et al., Science 1997;276:1268). Some 8 to 14 percent of the transcriptome consists of novel transcripts, 44 to 46 percent consists of uncharacterized cDNAs, and 42 to 46 percent consists of transcripts of known genes. Figure 9.1 shows the distribution of transcripts among the three cell types analyzed. Some 12 to 19.5 percent of the transcriptomes are of cell-specific transcripts while approximately 6,000 transcripts are shared by the three types of cells. It is worth noting that the most abundant tag matched with three UniGene clusters while the second most abundant tag represented an uncharacterized cDNA and the third most abundant tag had no match in the SAGE-map database. We validated differential expression between the SAGE libraries by quantitative real-time polymerase chain reaction (QPCR) of randomly selected transcripts. Components of the protein biosynthetic machinery are highly expressed in Spga. In Spcy, transcription factors are abundantly expressed, with chromosome-remodeling genes and testis-specific genes prominent in Sptd. We used comparative analysis of SAGE and QPCR data to reveal the presence of alternatively spliced variants. The present study is the first global study of gene expression in mouse germ cells and demonstrates that a core set of genes may be required for basic biological activities in all germ cells. However, cell-specific stages of spermatogenesis (mitotic, meiotic, and postmeiotic) require the concerted action of distinct sets of genes.
Pang A LY, Taylor HC, Johnson W, Alexander S, Chen Y, Su YA, Li X, Ravindranath N, Dym M, Rennert OM, Chan WY. Identification of differentially expressed genes in mouse spermatogen-esis. J Andrology 2003;24:59-71. Molecular cloning and characterization of genes in spermatogenesis Pang, Johnson, Ruszczyk, Baxendale, Rennert, Chan; in collaboration with Dym, Ravindranath, Su One major drawback of studies of gene expression during spermatogenesis is the lack of information on the expression of "housekeeping" genes in somatic tissues. Several reports indicate that the expression levels of some such genes would be altered under different physiological conditions, e.g., during cellular differentiation or when cells become malignant. We used QPCR analysis to examine the expression levels of 20 genes considered to be housekeeping genes in various studies of type A spermatogonia, pachytene spermatocytes, and round spermatids. Interestingly, expression levels of housekeeping genes such as glyceraldehyde-3-phosphate dehydrogenase, beta-tubulin 5, and cyclophilin D varied significantly among the three types of germ cells, whereas histone deacetylase 2 and 18S rRNA showed the least fluctuation in expression levels. This is the first study of housekeeping genes in germ cells; the data could serve as a standard for gene expression studies in distinct types of germ cells when precise gene expression measurements among these cells is needed. It has been reported that aberrant expression of some X-linked genes would cause failure in spermatogenesis as well as improper sexual development. A recent report indicated that spermatogonia express a substantial number of X-linked genes related to spermatogenesis. We have identified several X-linked genes that escape transcription inactivation at meiosis and in the postmeiotic stage or are expressed immediately before the pachytene stage. To gain a better understanding of the regulation of X-linked gene expression, we clustered the expression of all X-linked genes identified in germ cells. Two such genes, the Testis expressed gene 13 (Tex13) and Sex comb on midleg-like 2 (Scml2), were expressed predominantly in type A spermatogonia. In silico studies indicated the existence of a potential antisense transcript of Tex13, which is complementary to the 3' end of the Tex13 sense transcript. Reverse transcription-polymerase chain reaction (RT-PCR) studies revealed that the "trend of change" in expression of the sense and antisense transcripts was similar, but relative expression levels of the transcripts were dissimilar in germ cells at different stages. Regulation of the expression, biological activities, and relationship between the sense and antisense transcripts of Tex13 are currently under study. Scml2 is a member of the Polycomb group of genes, which encode transcriptional repressors essential for appropriate development in the fly and in mammals. We found at least three different Scml2 transcripts in mouse testes, each with a different 5' untranslated region, and observed alternative use of exons in the coding region. Using RT-PCR analyses, we found that the various forms of Scml2 transcripts were expressed at different levels during spermatogenesis, implying preferential use of the transcripts during germ cell development. In vivo as well as in vitro gene knock-down experiments are planned to study the functional roles of these X-linked genes during spermatogenesis. Using SAGE and cDNA microarray experiments, we cloned and characterized several novel as well as uncharacterized cDNAs identified as differentially expressed in germ cells. In addition, using a cDNA library screening and a 5' RACE, we cloned a novel transcript of about 1.1kb, shown to be preferentially expressed in round spermatids; we also cloned another spermatid-specific expressed sequence tag (EST) identified by cDNA microarray experiment. SAGE analyses of germ cell transcriptomes identified a number of novel transcripts, the more abundant of which are being cloned by RT-PCR with extended primers. Characterization and developmental studies of these cDNAs are expected to yield information on their roles in spermatogenesis. Blomberg LA, Chan WY, Clerch L, Massaro G, Massaro D. Molecular cloning and characterization of two genes up-regulated early in lung development. Biochim Biophys Acta 2002;1574:391-398. Functional genomic studies of gonad and early germ cell development Vong, Baxendale, Pang, Ruszczyk, Rennert, Chan; in collaboration with Culty, Lau Sexual dimorphism of the mouse gonad begins with expression of the Sry gene at around E11 (fetal day 11). When the fetal gonad is developing, primordial germ cells are moving and by E13 arrive at the genital ridge. Differentiation of the male and the female gonads and the concomitant development of the gonocytes and oocytes involve the intricate interaction of a large number of genes; the identity of the majority of these genes is still largely unknown. We have undertaken functional genomic studies using both SAGE and oligonucleotide microarray hybridization to delineate this phenomenon. Sexing of early gonads was done by RT-PCR to detect the expression of Sry while that of later gonads was achieved by morphological markers. A number of gonads and mesonephros were pooled for RNA extraction. We examined gene expression in the earlier gonads, i.e., E10.5, E11.5, and E12.5, by SAGE and that of the later dates, i.e., E13.5, E15.5, and E17.5 with the Affymetrix oligonucleotide GeneChip microarray. We also used SAGE to examine genes expressed in gonocytes harvested from newborn male mice. We are conducting analyses of the pattern of gene expression at different stages during development and are performing comparative analyses of gene expression in the male and female gonads. We compared the transcriptome of the gonocyte with that of type A spermatogonia as well as with that of E13.5 male gonads to delineate the group of gene clusters that regulate differentiation of primordial germ cells to gonocyte and then to spermatogonia. Results obtained from the studies are expected to yield information important for our understanding of the genetic regulation of gonad development and stem cell differentiation. Chan WY, Rennert OM. Molecular aspects of sexual differentiation. Curr Mol Med 2002;2:25-37. Physiological and genetic effects of disease-causing mutations of the luteinizing hormone receptor Leung, Baxendale, Wu, Aziz, Pang, Rennert, Chan; in collaboration with Al-Muslim, Fechner, Leschek, Martin Constitutive activating mutations of the human luteinizing hormone/chorionic gonadotropin receptor (hLHR) cause familial male-limited precocious puberty (FMPP), a noncentral form of gonadotropin-independent precocious puberty. An activating mutation leads to elevated production of testosterone and results in precocious puberty. All the activating mutations of the hLHR resulting in FMPP identified by others as well as by our laboratory are located in the transmembrane domain of the hLHR. Transmembrane helix VI (TMVI) is a particular hot spot of mutation. In a patient with FMPP, we recently identified the novel heterozygous mutation A1738G, which caused the replacement of Thr-580 by Ala. This mutation is downstream of the most frequently found activating mutation Asp578Gly. Our studies of transient transfection and signal transduction via the cAMP pathway as well as the inositol phosphate pathway demonstrated that Thr580Ala did not display the activation phenotype. The influence of the ligands of other G protein-coupled receptors such as FSH and TSH were likewise found to have no effect on signaling in cells expressing the mutated receptor. The results of the study argue for caution in the interpretation of genotype-phenotype correlations in hLHR mutations and FMPP. In collaboration with Malcolm Martin and Ellen Leschek, we identified two FMPP patients who developed testicular neoplasia. To study the potential tumorigenic effect of a constitutively activated LHR, we have generated an in vitro cell model and are in the process of generating a transgenic animal model. We transfected MA-10 cells with LHR carrying activating mutations. Using gene arrays on glass slides containing 22,000 mouse cDNAs, we compared the profile of expressed genes in cells expressing the mutated LHR with that of control cells. Preliminary studies of one mutated LHR indicated up-regulation of genes associated with cell proliferation and down-regulation of genes associated with differentiation. Interestingly, several genes known to be involved in spermatogenesis were also down-regulated in cells expressing the mutated LHR. We plan to compare the profile of gene expression affected by the more common activating mutation Asp578Gly with that affected by the somatic activating mutation Asp578His found in some patients with testicular tumors. The antithesis of FMPP is Leydig cell hypoplasia (LCH). In LCH patients, mutation inactivates the LHR, resulting in reduced production of testosterone and causing hypergonadotrophic hypogonadism or male pseudohermaphroditism. A novel missense mutation A340T resulting in the substitution of Ile-114 by Phe, which affects one of the six Leu repeats in the extracellular domain of hLHR receptor, has been identified in a patient with LCH. In transient expression studies, the mutant receptor failed to trigger cAMP production upon hCG stimulation. It is known that the mutated LHR, whether activated or inactivated, is abnormally processed by cells. To investigate the trafficking of the mutated LHR in vitro, we fused the coding sequence of green fluorescent protein (GFP) to that of wild-type and mutated LHR. We studied the trafficking of the fused protein by fluorescence microscopy. Information generated should further our understanding of the cellular processing of the LHR. The impact of activating mutation of the LHR has always been considered to be limited to the sexual development of a patient. The abnormal social behavior was thought to be secondary to precocious sexual maturation. Expression of LHR in brain had been demonstrated. We thus speculate that the abnormal behavior of FMPP patients is caused by the expression of the mutated LHR in the brain. To examine this hypothesis, we will identify the cellular location of LHR in the brain. We generated transgene cassettes with an open reading frame encoding a fusion protein of enhanced green fluorescent protein (EGFP) and hLHR mutant with substitution Asp578Gly or Asp578His. The expression of the fusion protein is driven by a 2.1 kb mouse LHR 5' flanking sequence. We will use the transgene cassettes to generate transgenic mice and then apply the animal model to studying the impact of constitutively activated LHR on spermatogenesis as well as on sexual and neurological development. Leung MLY, Al-Muslim O, Wu SM, Aziz A, Inam S, Awadh M, Rennert OM, Chan WY. A novel missense homozygous inactivating mutation in the fourth transmembrane helix of the luteinizing hormone receptor in Leydig cell hypoplasia. Am J Med Genet 2003; in press. Identification of genetic susceptibility to thrombosis in pseudotumor cerebri Dogulu, Baxendale, Leung, Chan, Rennert; in collaboration with Kansu, Ozguc Idiopathic intracranial hypertension (IIH), also known as pseudotumor cerebri (PTC), is a syndrome characterized by symptoms and signs of isolated intracranial hypertension. Even in the absence of clinical, laboratory, or radiological evidence of a space-occupying lesion or hydrocephalus, IIH can lead to catastrophic effects on the visual system. The clinical picture is caused by increased cerebrospinal fluid (CSF) pressure; however, its pathogenesis is not well understood. The most prevalent hypothesis is that the increased CSF pressure is attributable to reduced CSF absorption through the arachnoid villi. Although the majority of cases are nonfamilial, reports of familial PTC raise the possibility of genetic predisposition factors that lead to clinical manifestations following exposure to a precipitating agent. Thus, we hypothesize that IIH is multifactorial and that an underlying genetic thrombotic risk factor predisposes patients to develop local thrombi in the lining of the arachnoid villi, which in turn lead to increased intracranial pressure in the absence of demonstrable cerebral venous thrombosis as judged by conventional imaging techniques. Such a genetic "variation" might occur in coagulation factor V, an enzyme cofactor with pivotal functions in hemostasis. Several polymorphisms/mutations have been identified among the 25 exons of the Factor V gene. Altered activity of mutated Factor V is the most common hereditary blood coagulation disorder predisposing to thrombosis. Several mutations/polymorphisms of the Factor V gene, including VHong Kong (Arg306Gly), VCambridge (Arg306Thr), Arg485Lys, VLeiden (Arg506Gln), and the R2 allele (Arg-1299), all known to be associated with thrombotic risk, are located in exons 7, 10, and 13. We scanned the three exons for polymorphisms in 51 IIH patients and 68 controls. The prevalence of three FV thrombosis-associated polymorphisms (Factor VLeiden, 1628 G —>A substitution, and R2 allele) in IIH patients was found to be significantly higher than in controls, as was the incidence of associated prothrombotic states. Susceptibility to thrombosis may be an important genetic modifier, which, when present in combination with the other factors, is permissive for development of IIH. Our study is the first report of a genetic polymorphism related to an increased risk of IIH. Dogulu CF, Kansu T, Leung MYK, Baxendale V, Wu SM, Ozguc M, Chan WY, Rennert OM. Evidence for genetic susceptibility to thrombosis in Idiopathic Intracranial Hypertension. Thrombosis Res 2003; in press. Identification of the role of susceptibility to thrombosis in pseudotumor cerebri of nephropathic cystinosis Dogulu, Raygada, Chan, Rennert; in collaboration with Gahl, Kaiser In view of our recent findings regarding genetic susceptibility to thrombosis in PTC in general, we are studying the role of thrombosis susceptibility in the development of PTC in nephropathic cystinosis patients. We are screening nephropathic cystinosis patients who developed PTC and control nephropathic cystinosis patients without PTC by using a thrombosis susceptibility screening panel that includes PT, APTT, Activated Protein C resistance, serum levels of protein C, protein S, antithrombin III, fibrinogen, Factor VIII, Factor IX, Factor XI, total homocysteine, and antiphospholipid antibodies (ACA and Lupus AC). We are also screening for the FV Leiden mutation, FV G1628A polymorphism, FV R2 allele, Prothrombin 20210 mutation, and 5,10-methylenetetrahydrofolate reductase (MTHFR) gene C677T polymorphism in patients with severe homocysteinemia (e100 mol/l). Microarray analysis of the tumor suppressor effects of p75NTR and U94 in human prostate cancer Pang, Rennert, Chan; in collaboration with Ifon, Ravindranath, Rosenthal Nerve growth factor (NGF) binds to high-affinity tyrosine kinase receptors (TrkA, TrkB, or TrkC) and a low-affinity neurotrophin receptor (p75NTR) on the cell surface. In normal prostate epithelial cells, the major receptors for NGF are the TrkA form of the tyrosine kinase receptor and the p75NTR. During the development of prostate carcinoma, p75NTR was found to be progressively lost. The androgen-refractory human prostate cancer cell line PC3 also lacks the expression of p75NTR while TrkA expression is retained. Reintroduction of p75NTR into PC3 cells decreased the cells' tumorigenic ability in the nude mouse model. Neelakanta Ravindranath hypothesizes that p75NTR acts as a tumor suppressor by specifically regulating the expression of cancer-related genes. To identify the genetic network mediating the tumor suppressor effect of p75NTR, RNA samples isolated from PC3 cells stably expressing p75NTR or transfected with the vector alone were used in microarray hybridizations with human 6K cDNA microarrays to examine the global change in gene expression in the presence of p75NTR. A major portion of the genes showing differential expression patterns in the presence of p75NTR encode signaling proteins, together with growth factors and proteins involved in cell-cell contact. Leonard Rosenthal and Ekwere Ifon demonstrated that U94, a 1437 bp gene of human herpes virus 6A, can drastically inhibit foci formation by PC3 cells in vitro and inhibit the tumorigenicity of PC3 cells in the nude mouse model. The molecular mechanism underlying such phenotypic changes remains largely unknown. We used a functional genomic approach similar to that described above to examine the genes that are expressed differentially in the presence of U94. Using the human 6K cDNA microarrays, we subjected RNA isolated from the PC3 cells transfected with or without U94 to cDNA microarray analysis and found that expression of specific sets of genes related to cell-cell contact, proliferation, oncogenes, and growth factor receptors were differentially modulated in the presence of U94. COLLABORATORS Osama Al-Muslim, MD, Riyadh Armed Forces Hospital, Riyadh, Saudi Arabia Martine Culty, PhD, Georgetown University, Washington DC Martin Dym, PhD, Georgetown University, Washington DC Patricia Fechner, MD, Stanford University, Palo Alto CA William Gahl, MD, PhD, Medical Genetics Branch, NHGRI, Bethesda MD Ekwere Ifon, PhD, Georgetown University, Washington DC Muriel I. Kaiser-Kupfer, MD, Ophthalmic Genetics and Clinical Services Branch, NEI, Bethesda MD Tulay Kansu, MD, Hacettepe University, Ankara, Turkey Chris Y.F. Lau, PhD, University of California, San Francisco CA Ellen W. Leschek, MD, Developmental Endocrinology Branch, NICHD, Bethesda MD Malcolm M. Martin, MD, Georgetown University, Washington DC Meral Ozguc, PhD, Hacettepe University, Ankara, Turkey Neelakanta Ravindranath, PhD, Georgetown University, Washington DC Leonard Rosenthal, PhD, Georgetown University, Washington DC Yan Su, MD, PhD, Loyola University, Chicago IL Timothy Stitely, MS, Unit on Computer Support Services, NICHD, Bethesda MD
For further information, contact chanwy@mail.nih.gov |
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