Owen M. Rennert, MD, Head, Section on Developmental Genomics
Shao-Ming Wu, PhD, Staff Scientist
Margarita Raygada, PhD, Staff Genetic Counselor
Alan L.Y. Pang, PhD, Senior Fellow
Maria Grekova, MD, PhD, Research Fellow
Martin Dym, PhD, Visiting Scientist
Cigdem F. Dogulu, MD, PhD, Clinical Fellow
Laszlo Horvath, PhD, Postdoctoral Fellow
Tin-Lap Lee, PhD, Postdoctoral Fellow
Xingli Meng, MD, PhD, Postdoctoral Fellow
Wai-Yee Chan, PhD, Adjunct Investigator
Diana Alba, BS, Postbaccalaureate Fellow
Elaina Mikhaylova, BS, Postbaccalaureate Fellow
Stephanie Peacock, BS, Postbaccalaureate Fellow
Lisa Ruszczyk, BS, Postbaccalaureate Fellow
Vanessa Baxendale, MS, Research Associate

We apply system biology approaches to study the regulation of gene expression and function in the developmental process in health and disease. Normal development is dictated by intricate relationships among a multitude of genes and by interactions of several biological pathways. We apply functional genomic approaches and related technologies to study gene expression and action in the well-characterized process of murine embryonic gonad development and spermatogenesis. At different stages of development, mouse embryonic gonads and male germ cells have distinct morphological features and genetic markers that allow their identification. Consequently, characterization of stage-specific gene expression and biological pathways can be achieved for both mouse embryonic gonad development and spermatogenesis. The goal of our research is to delineate the network of genes that regulate renewal and differentiation of spermatogonial stem cells, meiosis, and the post-meiotic differentiation of germ cells and to develop novel methods of fertility control and treatment for the infertile. Related to our work on mouse development is our study of the role of luteinizing hormone/choriogonadotropin receptor (hLHR) in human development and diseases. To date, our research has revealed a novel role of hCG in neuronal development. Expression profiling has disclosed distinct genes triggered by two mutants of hLHR.

Identification and functional characterization of spermatogenesis stage-specific genes

Pang, Peacock, Rennert, Chan

Based on expression analysis of mouse type A spermatogonia, pachytene spermatocytes, and round spermatids, we cloned a novel mArd1 (Arrest Defective 1) homologue, which we named mArd2, that demonstrated testis specificity and elevated expression in pachytene spermatocytes. The mArd1 protein is known to interact with an auxiliary protein subunit mNAT1 to constitute a functional N-acetyltransferase. Earlier studies in yeasts identified diverse roles for ARD1, ranging from cell cycle regulation to DNA repair and recombination. We showed that the transcript of mArd2 is preferentially expressed in male meiotic germ cells while the expression of the encoded protein is delayed. By performing an in vitro protein pull-down assay and an N-acetyltransferase activity assay, we demonstrated that Ard2, like Ard1, could interact with Nat1 and display N-acetyltransferase activity. Our data imply that Ard1 and Ard2 are functionally homologous. The expression of Ard2 may therefore be responsible for compensation of the loss of X-linked Ard1 starting from meiosis. An isoform of Ard1 has been shown to acetylate the ε-amino group of Hif-1α. We are now testing whether Ard2 would display such activity, i.e., altered substrate specificity, in germ cells. We are also examining the effect of promoter elements and DNA methylation on transcriptional activation of Ard2 during spermatogenesis.

Another gene that we identified by expression profiling of male germ cells is mLin28, a heterochronic gene whose product regulates developmental timing in C. elegans. It has been suggested that Lin28 protein regulates the decision between cellular proliferation and differentiation. However, the biochemical properties of Lin28 are not well studied. We observed differential use of transcription start sites of mLin28 transcripts as germ cells differentiate and identified specific promoter elements and modules that were known to elicit transcriptional activation effect. The 3′ end of mLin28 transcripts was heterogeneous; we had isolated alternative 3′ ends of the transcripts, which displayed stage-specific expression patterns. To understand more fully the regulation of Lin28 expression, we are analyzing the gene's promoter activity. We also generated a P19 cell line stably expressing an HA-tagged version of Lin28 for the identification of proteins and RNA molecules that may interact with Lin28 and regulate its production. Transcript knockdown experiments with siRNA against Lin28 are in progress to study the function of Lin28 protein in vitro.

In addition to mArd2 and mLin28, we studied the expression of Ddx3y (formerly known as Dby), a Y-encoded gene, during male gonad development and spermatogenesis. Our studies indicated that, in accord with meiotic sex chromosome inactivation, Ddx3y and its X chromosome homologue Ddx3 are replaced in spermatocytes by their autosomal retroposon D1Pas1 (also known as PL10). Unlike DDX3Y in human, the role of Ddx3y in spermatogenesis in the mouse is less obvious, and its biological activity may be replaced by that of Ddx3 and its autosomal homologue.

Characterization of selected genetic processes in spermatogenesis

Lee, Wu, Alba, Ruszczyk, Mikhaylova, Horvath, Baxendale, Rennert, Chan

We established an expression database of mouse male germ cells through the use of Serial Analysis of Gene Expression (SAGE). Computational analyses had led to the identification of stage-specific pathways and promoter modules and the construction of biological networks associated with different stages of spermatogenesis. In addition, these analyses allowed us to identify a large number of genes with stage-specific alternative-spliced variants. It has been suggested that alternative splicing is a prominent genetic process during spermatogenesis. A number of genes have been known to undergo alternative splicing, which confers novel activities to the variants. However, no systematic study has investigated the stage specificity of the splicing mechanism and the expression of the variants. We have initiated the characterization of novel stage-specific variants of a number of genes, including heat shock protein 4 (Hspa4), H3 histone, family 3B (H3f3b), and ubiquitin protein ligase E3A (Ube3a). We will use Hspa4 as a model to investigate the role of alternative splicing in stage-specific regulation of gene function and its impact on the biological activity of the splice variants. Hspa4 has been shown to be induced in response to oxidative stress, which is critical for the survival and normal functioning of spermatozoa and male fertility. We confirmed the presence of three distinctive transcripts of Hspa4 in type A spermatogonia, pachytene spermatocytes, and round spermatids. Further biochemical and functional studies are under way to characterize Hspa4's regulatory mechanisms and the biological functions of the isoforms in germ cells.

Analysis of the germ cell SAGE database also revealed the prominent presence of antisense transcripts. We are particularly intrigued by the presence of antisense transcripts derived from pseudogenes. We identified 19 genes with antisense transcripts; of these, four (Uba52, Ch10, Calm2, and Ubb) had antisense transcripts derived from their pseudogenes on different chromosomes. Apparently, these pseudogenes were derived from reverse transcripts of the respective parent genes and transposed to the intron of actively transcribed genes: the Uba52 pseudogene resides in the intron of Cbx1, that of Calm2 in the intron of Prkar2b, that of Ch10 in the intron of Sp3, and that of Ubb in the intron of Catsper2. More interestingly, the orientation of the pseudogenes is anti-parallel to that of their host genes. Thus, the antisense transcripts of the pseudogenes would be produced as processed introns of their respective host genes, raising the possibility that the two anti-parallel transcription units interact through hybridization of the sense-antisense transcripts. Subsequent experiments confirmed the presence of native double-stranded RNA of the anti-parallel genes, namely, Uba52-Cbx1, Ch10-Sp3, and Calm2-Prkar2b. We have not yet confirmed the presence of double-stranded RNA of Ubb-Catsper2. We will examine the relationship between the anti-parallel gene pairs by using Uba52 and Cbx1 as a model. The functional gene of Uba52 is on chromosome 8 while its pseudogene is on chromosome 11, embedded in the first intron of Cbx1. Uba52, Cbx1, and the sense and antisense transcripts of the Uba52 pseudogene are expressed in mouse kidney cell line CRL-6436, which we will use as a model for the study.

Functional genomic studies of male gonad development

Lee, Alba, Rennert, Chan; in collaboration with Lau, Li

Despite the identification of key genes, such as Sry, that are integral during embryonic gonad development, the mechanisms that regulate such critical biological processes, such as transition of the primordial germ cells to gonocytes and the initiation of sexual dimorphism, are still far from understood. Using SAGE, we are attempting to delineate the transcriptome of male embryonic gonads at different stages of development. We profiled the genes and novel transcripts expressed in male mouse embryonic gonads at E10.5 (embryonic day 10.5), E11.5, E12.5, E13.5, E15.5, and E17.5 with an average of 152K coverage for each of the SAGE libraries. The six transcriptomes gave a total of about 45,000 known transcripts, about 21,000 uncharacterized cDNAs, and about 4,000 novel transcripts. Among the 10 most abundantly expressed transcripts, five encoded ribosomal proteins similar to those observed in human embryonic stem cells. Six of the most abundantly expressed transcripts, including cytochrome b oxidase subunit 5b (Cox5), cytochrome b-245 beta polypeptide (Cybb), tumor protein, translationally controlled 1 (Tpt1), ribosomal protein L19 (Rpl19), ubiquitin A-52 residue ribosomal protein fusion product 1 (Uba52), and an uncharacterized cDNA, were also among the 10 most abundant transcripts in type A spermatogonia. Application of unsupervised clustering analysis to the embryonic gonad transcriptomes yielded distinct genomic fingerprints of two major stages in gonad development, namely, early (E10.5-E12.5) and late (E13.5-E17.5) stages.

Figure 9.1

To identify the chromosomal regions most active during gonad development, we created algorithms to assign the SAGE tags to the corresponding chromosomal position based on the Unigene assignments. We then displayed the tags in chromosome graphic format by using the completed mouse genome information. Consequently, we created a chromosome heatmap (see Figure 9.1) of embryonic gonad development that, based on co-localization of gene expression, highlighted chromosomal "hotspots" that exhibited significant changes at any time point of development. The chromosome heatmap will be useful to determine the importance of different regions at specific time points during developmental process as well as to identify the potential consequences of chromosome duplication or deletion. Furthermore, the heatmap will permit extraction of the biological implications of significant chromosomal clusters and further analysis by linking to different mutation and functional annotation databases, including Online Mendelian Inheritance in Man (OMIM) and EASE.

Non-conventional activities of luteinizing hormone/choriogonadotropin receptor

Meng, Lee, Rennert, Chan; in collaboration with Fechner, Jeha, Karaviti, Leung, Steinbach

The luteinizing hormone/choriogonadotropin receptor (LHR) plays a central role in human male sexual development. Mutation of LHR results in abnormal production of testosterone and diseases of sexual development. Though the role of LHR in transducing the signal of luteinizing hormone/chorionic gonadotropin is well established, the mechanism of its action is still not fully understood. We have studied the molecular genetics of LHR from a large number of patients with activating and inactivating LHR mutations. The studies help us understand varied presentation of diseases as well as the molecular mechanism of the receptor's signal transduction.

Individuals with LHR that carries activating mutations develop familial male-limited precocious puberty (FMPP) and often exhibit behavioral problems. Such behavioral problems may be related to the dysfunction of brain cells caused by the mutated receptor. Given that the major growth period of mammalian brain occurs in the first trimester of pregnancy when hCG is high and LHR is expressed in brain cells, it has long been suspected that the hCG-LHR pathway is involved in the development of the early brain. We attempted to assess the activity of LHR in brain cells by using PC12, a rat neuronal cell line that is bipotent and can differentiate into either neuronal or chromaffin cells upon exposure to neurotropins or differentiating inducing agents. PC12 cells expressing hLHR carrying the activating mutation Asp578His demonstrated neurite outgrowth in about 11 percent of cells, significantly higher than that of cells transfected with vector or wild-type hLHR. The addition of hCG to the culture medium of stably transfected cells expressing wild-type LHR caused significantly more neurite-bearing cells than in no-hCG treatment controls or vector-expressing controls. The effect of hCG was dose- and time-dependent. Differentiated cells appeared as early as 24 hours after hCG administration and demonstrated profound morphological changes by 72 hours. The differentiated cells expressed early neuronal markers, neuronal-specific tubulin III, and neural filament 68, indicating that LHR activation—through either genetic mutation of the receptor itself or binding with its ligand—induced the differentiation of PC12 cells toward neuronal cell type. Further studies showed that both the p44/42 and p38 pathways were required for the neuronal differentiation of PC12 cells transfected with wild-type hLHR and induced by hCG while the SAPK/JNK MAPK and Akt pathways might not be involved. cAMP played an important role in transmitting signals from receptor activation to these signaling pathways. Our results demonstrate a neural function of the hCG/LHR pathway and show the neurotropic activity of hCG and its potential as a therapeutic agent for neurological disorders and acute injuries of the nervous system.

Discovery of the presence of LHR with germline and somatic activating mutations in patients with testicular tumor raised the question of the tumorigenic potential of mutated LHR. Asp578His is a somatic mutation; hLHR carrying the mutation has been found only in testicular tumor tissues and not in any patient with FMPP. On the other hand, Asp578Gly is the most common mutation detected in FMPP patients and can be transmitted through the germline. Animal studies have so far failed to establish lines of male or female transgenic founder mice carrying LHR with the Asp578His mutation. We speculate that, even though Asp578Gly and Asp578His involve mutation of the same amino acid, the two mutant hLHRs have distinct biological effects and trigger expression of different sets of genes. To prove our hypothesis, we compared the expression profile of MA10 cells transfected with mutated LHR carrying the germline activating mutation (Asp578Gly) with those expressing the somatic activating mutation (Asp578His). Results revealed different expression patterns as a consequence of the expression of LHR with the Asp578Gly or Asp578His mutation. We are in the process of delineating the biological pathways affected by these mutant LHRs.

Genomic and genetic studies of heritable disorders

Rennert, Chan, Raygada, Dogulu

One of the missions of the Laboratory of Clinical Genomics (LCG) is to train physicians in the application of genomic and genetic approaches in studies of human diseases. Accordingly, we attempt to devise global approaches for identifying and screening risk factors of complex disorders. We have designed two hybridization-based high-throughput methods for screening susceptible risk factors for thrombophilia and age-related macular degeneration. In addition to undertaking basic and translational research, LCG has developed clinical protocols to study patients with genetic and metabolic disorders, thereby providing us with access to various genetic disorders and enabling us to deliver genetics training to our fellows.

Application of high-throughput approaches in the study of complex disorders

Dogulu, Chan, Rennert; in collaboration with Su

Venous thrombosis affects 1 in 1,000 individuals annually and is one of the leading causes of mortality and morbidity, resulting in approximately 300,000 hospitalizations and 50,000 fatalities per year in the United States alone. It is, however, an avoidable disease if currently available prophylactic treatment is instituted. Our calculations demonstrated that concurrent use of a panel of 10 genetic tests increases the positive predictive value of testing for venous thrombosis at least 25-fold. We have devised an approach (Method Evolved for Recognition of Thrombophilia [MERT], patent pending) that will allow prediction and accurate assessment of hereditary thrombophilia in several ethnic populations by rapid, concurrent screening of an array of all known 143 venous thrombosis-associated recurrent mutations and polymorphisms in eight genes. MERT will help us develop stratification protocols for risk-adapted prophylaxis. We obtained positive results in preliminary studies and are now in the process of verifying the clinical applicability of the fabricated microarrays.

We applied a similar approach to the design of a microarray for screening for susceptibility to development of age-related macular degeneration (Method Evolved for Recognition and Testing of Age-Related Macular Degeneration-[MERT-ARMD]). Age-related macular degeneration (ARMD) is the most common cause of severe vision loss in the United States and developed countries among people 65 years of age and older. It has been suggested that ARMD is a multifactorial disorder. Our previous reports described screening for one or more polymorphisms associated with ARMD. In general, the use of these assays is limited because they have a low predictive value and detect mutations prevalent only in Caucasian populations. We have designed a MERT-ARMD that will concurrently screen 90 known age-related macular degeneration-associated mutations and polymorphisms in 16 molecules, using hybridization-based, high-density oligonucleotide array technology.

Identification of the role of susceptibility to thrombosis in pseudotumor cerebri of nephropathic cystinosis

Dogulu, Raygada, Chan, Rennert; in collaboration with Gahl, Kaiser

Given our findings regarding genetic susceptibility to thrombosis in pseudotumor cerebri (PTC), we are studying the role of thrombosis in the development of PTC in nephropathic cystinosis. Using a thrombosis susceptibility panel, we are screening nephropathic cystinosis patients who develop PTC as well as control nephropathic cystinosis patients without PTC. The panel includes prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT), activated protein C resistance (APCR), serum levels of protein C and S, antithrombin III, fibrinogen, total homocysteine, antiphospholipid antibodies (ACA panel and Lupus AC). In patients with severe homocysteinemia (greater than or equal to 100 micro mol/l), we are screening for the FV Leiden mutation, FV G1628A polymorphism, FV R2 allele, prothrombin 20210 mutation, and 5,10-methylenetetrahydrofolate reductase (MTHFR) gene C677T polymorphisms. To date, we have recruited five patients with PTC with pre-existing nephropathic cystinosis. The thrombosis screening panel revealed shortened thrombin time (TT) in two patients, high-titer anticardiolipin (ACA) IgM antibodies in one patient, and activated protein C resistance (APCR) in one patient. Thrombin time measures the rate of fibrin monomer polymerization and is the most sensitive screening test for decreases or abnormalities in fibrinogen (a shortened TT demonstrates an acceleration of fibrin monomer polymerization, which contributes to thrombotic tendency). Activated protein C resistance is a condition that leads to a hypercoagulable state with an increased risk for venous thrombosis; the IgM isotype of ACA has been shown to be associated with venous thrombosis.

Clinical protocol on the studies of patients with genetic and metabolic disorders

Raygada, Dogulu, Rennert; in collaboration with Kaler, Stratakis

Of the two active protocols in our program, one aims at providing care for patients with a variety of rare genetic disorders, supplementing and offering additional training opportunities in clinical genetics, dysmorphology, and metabolic genetics in NICHD and other institutes of the NIH. We examine patients with a broad spectrum of metabolic and genetic conditions and have thus far evaluated 386 individuals. In addition, we offer genetic counseling services to patients and their families to assess risk, and we provide information on preventive measures and testing options. Disorders of interest include chromosomal and Mendelian disorders of childhood and/or adult onset, congenital anomalies and/or birth defects, dysmorphic syndromes, familial cancer syndromes, multifactorial disorders, and metabolic abnormalities. If not eligible for another NICHD research protocol (specific for a disease or treatment), patients with genetic/metabolic-related conditions may be evaluated under the auspices of our protocol to advance the clinical skills of physicians participating in NICHD clinical research and training programs, thereby providing an impetus for clinical research initiatives. Standard, medically indicated laboratory or radiological studies may be performed to confirm a diagnosis or to aid in disease management. In some cases, the patient receives medical or surgical treatment according to current clinical practice. Patients and/or family members with genetic disorders may offer their DNA for storage and/or testing.

The second of the two protocols aims at unraveling the contributions of insulin and insulin-related actions (e.g., insulin resistance, abdominal fat) to breast cancer risk. The ultimate goal is to develop new strategies for risk reduction and early screening of at-risk patients. We will determine whether factors associated with or governing insulin function may be involved in modification of breast cancer risk in specific patient populations. We are working to determine the role of abdominal fat and other anthropometric measures, as well as their interaction with insulin and other hormones in breast cancer risk in patients with and without a positive family history (classified by menopausal status). We hope to obtain sufficient data for future studies that look at mediators of the actions of insulin on breast cancer (IRS-1, IRA isoform) and the gene polymorphisms involved in a specific patient population. Finally, we plan to develop rational, cost-efficient guidelines for risk-reducing and screening strategies for a subset of patients responsive to the growth-promoting actions of insulin. We began patient recruitment in September 2006.

COLLABORATORS

Patricia Fechner, MD, Stanford University, Palo Alto, CA
William Gahl, MD, PhD, Clinical Director, NHGRI, Bethesda, MD
George Jeha, MD, Baylor College of Medicine, Houston, TX_
Muriel I. Kaiser, MD, Ophthalmic Genetics and Visual Function Branch, NEI, Bethesda, MD
Stephen Kaler, MD, MPH, Laboratory of Clinical Genomics, NICHD, Bethesda, MD
Lefkothea P. Karaviti, MD, Baylor College of Medicine, Houston, TX
Chris Y.F. Lau, PhD, University of California San Francisco, San Francisco, CA
Michael Yiu-Kwong Leung, PhD, Rush College of Medicine, Chicago, IL
Yunmin Li, PhD, University of California San Francisco, San Francisco, CA
Peter Steinbach, PhD, Center for Molecular Modeling, CIT, NIH, Bethesda, MD
Constantine Stratakis, MD, DSc, Heritable Disorders Branch, NICHD, Bethesda, MD
Yan Su, MD, PhD, Loyola University, Chicago, IL

For further information, contact rennerto@mail.nih.gov.