Genetic and Genomic Studies in Normal Development and Diseases

Owen M. Rennert, MD, Head, Section on Clinical Genomics
Alan L.Y. Pang, PhD, Biologist
Margarita Raygada, MS, PhD, Staff Genetic Counselor
Cigdem F. Dogulu, MD, PhD, Clinical Fellow
Elizabeth Jiali Fang, Summer Student

We focus on translating genetic and genomic technologies, acquired through basic studies, into research on clinical problems. One of our missions is to train physicians in the application of genomic and genetic approaches to studies of human diseases. Using the cutting-edge methylation tiling array technology, we are studying the transcription regulation of mArd2, a novel testis-specific gene that was first cloned in our laboratory. We also study Lin 28, a gene that has been shown to regulate developmental timing. Proteomic approaches helped the identification of RNA s bound to the protein encoded by this gene. Gene knockdown experiments are under way to study the function of Lin 28 in determining cell fate. Relevant to our missions is our attempt to define global approaches for identifying and screening for the risk factors for complex disorders. To this end, we have designed two-hybridization–based high-throughput screening methods to detect susceptibility risk factors for thrombophilia and age-related macular degeneration. Besides basic and translational research, we are involved in clinical protocols to study patients with genetic and metabolic disorders, allowing us to deliver clinical genetics training to our fellows in what is a critical component of our work.

Transcription Regulation and Functional Studies of Testis-Specific Genes

Genetic, genomic, and proteomic studies of a testis-predominant isoform of a mouse acetyltransferase

Pang, Fang, Rennert; in collaboration with Chan, Taft

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 (also known as Ard1b), 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. We showed that the protein encoded by mArd2 is functionally similar to its homologue mArd1 in vitro. However, the two homologous genes displayed grossly different expression patterns in various cell lines and mouse tissues. Specifically, the mArd2 gene is expressed in a developmentally regulated manner during spermatogenesis. To elucidate the molecular mechanism of the gene’s tissue specificity, we performed an mArd2 promoter assay in two mArd2 non-expressing cell lines, namely, NIH/3T3 and GC-2spd(ts), which represent cells of somatic and early germline origin, respectively. The detection of mArd2 promoter activities in both cell types indicates that the transcriptional machinery required for mArd2 expression is intact; the absence of mArd2 expression would thus result from other regulatory mechanisms. We hypothesize that DNA methylation could play a role in the regulation of mArd2 expression.

Click for a larger version.
Figure 10.3
Methylation status of the two CpG islands spanning the proximal promoter and half of the coding region of the mArd2 gene in different tissues determined by bisulfite sequencing. Filled circles represent methylated cytosine and empty circles represent unmethylated cytosine. GC-2spd(ts) = spermatogonial cell line; SgA = spermatongonia; PcSc = pachytene spermatocyte; RdSd = round spermatid.

Using 5-aza-deoxycytidine treatment in nonexpressing cells, in vitro methylation, and bisulfite sequencing analysis, we showed that mArd2 transcription is regulated epigenetically. Comparison of the methylation status of the two CpG islands spanning the proximal promoter and half of the coding region of the mArd2 gene showed that its proximal promoter was hypermethylated in mouse tissues that do not express mArd2 but hypomethylated in male germ cells, which express the gene. On the other hand, we found that the distal CpG island was hypomethylated in germ cells at stages that display higher mArd2 expression but hypermethylated in non-expressing tissues as well as in mitotic germ cells, in which mArd2 expression is minimal. Thus, our findings confirmed the hypothesis that testisspecific expression of mArd2 is epigenetically regulated. The Ard1-Ard2 expression system could be a paradigm for studying epigenetic regulation of mammalian spermatogenic gene expression.

Despite the above findings, the involvement of other protein factors in the activation of Ard2 transcription therefore attempting to identify the minimal Ard2 promoter. Our is proximal to the transcriptional start site of the gene and overlaps islands. We are now isolating the factors that are bound to this sequence activation and suppression of Ard2 transcription is regulated.

We also demonstrated that the translation of Ard2 in vivo is delayed during spermatogenesis; the transcript level peaks in meiotic germ cells, but the protein is not expressed until after the appearance of round spermatids. We hypothesized that the 3′ untranslated region (UTR) of Ard2 confers a repressive effect on the gene's translation. To test the UTR’s ability to repress reporter gene expression, we established a cell culture model and identified several regions on the 3′ UTR that are involved in the suppression of reporter translation. We are isolating the testicular protein factors that would be bound to these sequences and will study the involvement of the factors in regulating the translation of other spermatogenic transcripts.

Using gene knockdown experiments in mammalian cells, we are also studying the global effect of a deficiency of protein N-alpha terminal acetylation on cell functions.

Examination of the biological role of Lin28 in mitotic male germ cells

Pang, Rennert; in collaboration with Chan, Taft

Lin28 is a heterochronic gene involved in the temporal control of cell fate determination in C. elegans. In mammals, Lin28 is detected mostly in cells that possess proliferative capacity, e.g., embryonic stem cells, embryonal carcinoma cells, and mouse type A spermatogonia. Lin28 is known to be localized predominantly in the cytoplasm. However, a recent study showed that Lin28 is involved in a microRNA maturation process in the nucleus, suggesting that the protein may play a dual role in the cell by interacting preferentially with nuclear and cytoplasmic proteins. However, the gene’s biological function, especially its role in mammalian development, remains largely unknown.

We hypothesized that Lin28 acts as an RNA chaperone to control the availability of transcripts that encode functions important to cell fate determination. In a cell culture model, we have identified a set of RNA transcripts that bind to Lin28; using gene knockdown, we are now studying the corresponding change in protein levels of the transcripts. We are also isolating the proteins that interact with Lin28 in the nuclear and cytoplasmic fractions of expressing cells in order to examine the protein’s biochemical role.

The selectivity of gene expression in certain cell types suggests that transcription of Lin28 is strictly regulated. Our preliminary data showed that DNA methylation does not regulate Lin28 transcription. Instead, a specific genomic region about 1 kilobp upstream of the transcriptional start site showed promoter activity only in Lin28-expressing cells. We are screening a panel of Lin28-expressing and -non-expressing cells to identify the genetic factors involved in activating or suppressing Lin28 transcription.

Chan WY, Wu S, Ruszczyk LM, Lee T, Rennert OM. Antisense transcription in developing male germ cells. In: Lau YFC, Chan WY, eds. Y Chromosome and Male Germ Cell Biology in Health and Diseases. World Scientific Publishers, 2007;201-220.
Pang ALY, Martin MM, Martin ALA, Chan WY. Molecular basis of diseases of the endocrine system. In: Coleman WB, Tsongalis GJ, eds. Molecular Pathology: the Molecular Basis of Human Disease. Elsevier-Academic Press, 2009, in press.
Pang AL Y, Peacock S, Johnson W, Bear DH , Rennert OM , Chan WY. Cloning, characterization and expression analysis of a novel acetyltransferase retrogene Ard1b in the mouse. Biol Reprod 2009, in press.

Genomic and Genetic Studies of Heritable Disorders

In pursuit of our goal to develop global approaches for identifying and screening for risk factors of complex disorders, we designed two-hybridization–based high-throughput methods that can screen for susceptible risk factors of thrombophilia and age-related macular degeneration. Besides basic and translational research, we have instituted clinical protocols to study patients with genetic and metabolic disorders.

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

Dogulu, Rennert; in collaboration with Chan, Su

Venous thrombosis (VT) 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, with an incidence of 141 per 100,000 African Americans, 104 per 100,000 Caucasians, 55 per 100,000 Hispanics, and 21 per 100,000 Asian/Pacific Islanders. Our calculations demonstrated that concurrent use of a panel of 11 genetic tests increases by at least 30-fold the positive predictive value of testing for VT. We have patented a Method Evolved for Recognition of Thrombophilia [ME RT] that is currently under evaluation for proof of principle for potential prediction and accurate assessment of hereditary thrombophilia in several ethnic populations. The methodology involves rapid, concurrent screening of an array of all known 145 venous thrombosis–associated recurrent mutations and polymorphisms in nine genes [antithrombin III (AT III), protein C, protein S, fibrinogen, factor V (FV), prothrombin (factor II), methylenetetrahydrofolate reductase (MTH FR), angiotensin 1–converting enzyme (ACE), and plasminogen activator inhibitor-1 (PAI-1)]. We have designed 291 oligonucleotide 25-mer probes to be spotted onto the microarray and, using Multiplex PCR assay, amplified 40 amplicons covering the variation sequences from nine genes in a single amplification reaction. We are now verifying our method’s analytic validity.

We have patented a similar approach for the design of a microarray screening for susceptibility to age-related macular degeneration (Method Evolved for Recognition and Testing of Age-Related Macular Degeneration [MERT-ARMD]). Among people 65 years of age and older, age-related macular degeneration (ARMD) is the most common cause of severe vision loss in the United States and developed countries. It is a multifactorial disorder. We have designed a MERT-ARMD that, using hybridization-based, high-density oligonucleotide array technology, will screen concurrently for 105 known age-related macular degeneration–associated mutations and polymorphisms in 16 genes (CFH, LOC387715, BF, C2, ABCR, Fibulin 5, VMD2, TLR4, CX3CR1, CST3, MnSOD, MEHE, paraoxonase, APOE, ELOVL4, and hemicentin-1).

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

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

In support of the Institute’s research mission, we are expanding the spectrum of diseases seen in our clinics and inpatient units, thereby broadening training opportunities for our clinical fellows. We are conducting two active protocols. Protocol # 02-CH-0023 provides diagnostic and consultative services to patients with a variety of rare genetic disorders; in addition, it supplements and offers additional training in clinical genetics, dysmorphology, and human biochemical genetics in NICHD and other Institutes. Under the protocol, we have evaluated more than 450 patients with a broad spectrum of metabolic and genetic conditions. For five years, we have accommodated pediatric residents from Georgetown University Hospital in our protocol. Starting in July 2003, the residents have rotated in our clinic every month. In addition, during the past year, we began the rotation of all Intramural Training awardees in NIH (three per week). We perform evaluations of local and out-of-state patients with suspected or diagnosed genetic/metabolic disorders; we have established a well-known resource for patients and physicians who need counseling and/or evaluation of these disorders. Clinical services include genetic counseling, risk assessment, diagnostic testing, and discussion of preventive interventions. We often perform standard, medically indicated laboratory or radiological studies to confirm a diagnosis or to aid in the management of patients. In some cases, patients receive medical or surgical treatment for their disorder, according to current clinical practice. Patients and/or family members with genetic disorders may offer their DNA for storage and/or testing.

Protocol #06-CH-0119 aims to unravel the contributions of insulin and insulin-related actions (e.g., insulin resistance, abdominal fat) to breast cancer risk, with the ultimate goal of early screening of at-risk patients and development of new strategies for risk reduction. Our study plan focuses on (1) determining whether factors associated with or governing insulin function may be involved in the modification of breast cancer risk in specific patient populations; (2) determining the role of abdominal fat and other anthropometric measures as related to, first, breast cancer risk in patients with and without a positive family history (classified by menopausal status) and, second, to interaction with insulin and other hormones; (3) providing sufficient data for future studies that investigate mediators of the actions of insulin on breast cancer (IRS-1, IRA isoform) and gene polymorphisms in this patient population; and (4) developing rational, cost-efficient guidelines for risk-reducing and screening strategies for a subset of patients responsive to the growth-promoting actions of insulin. As of this writing, we have evaluated more than 20 patients. The reduction in funds to cover patient costs has hindered patient recruitment. We are analyzing our pilot data and will subsequently determine any modifications that might be required for continuation of the protocol.

Bai X, Wu J, Zhang Q, Alesci S, Manoli I, Blackman MR, Chrousos GP, Goldstein AL, Rennert OM, Su YA. Third-generation human mitochondria-focused cDNA microarray and its bioinformatic tools for analysis of gene expression. Biotechniques 2007;42:365-375.
Manoli I, Alesci S, Blackman MR, Su YA, Rennert OM, Chrousos GP. Mitochondria as key components of the stress response. Trends Endocrinol Metab 2007;18:190-198.
Meng XL, Rennert OM , Chan WY. Human chorionic gonadotropin induced neuronal differentiation of PC12 cells through activation of stably expressed lutropin/choriogonadotropin receptor. Endocrinology 2007;148:5865-5873.
Ohta S, Lai EW, Morris JC, Pang ALY, Watanabe M, Yazawa H, Zhang R, Green JE, Chan WY, Sirajuddin P, Taniguchi S, Powers JF, Tischler AS, Pacak K. Metastasis-associated gene expression profile of liver and subcutaneous lesions derived from mouse pheochromocytoma cells. Mol Carcinog 2008;47:245-251.
Timmers H, Kozupa A, Eisenhofer G, Raygada M, Adams K, Solis D, Lenders J, Pacak K. Clinical presentations, biochemical phenotypes, and genotype-phenotype correlations in patients with Succinate Dehydrogenase Subunit B-associated pheochromocytomas and paragangliomas. J Clin Endocrinol Metab 2007;92:779-786.

Collaborators

Wai-Yee Chan, PhD, Program in Reproductive and Adult Endocrinology, NICHD, Bethesda, MD
Muriel I. Kaiser, MD, Ophthalmic Genetics and Visual Function Branch, NEI, Bethesda, MD
Stephen Kaler, MD, MPH, Program in Reproductive and Adult Endocrinology, NICHD, Bethesda, MD
Constantine Stratakis, MD, D(med)Sci, Program in Developmental Endocrinology and Genetics, NICHD, Bethesda, MD
Yan A. Su, MD, PhD, George Washington University, Washington, DC
Diana H. Taft, BS, Program in Reproductive and Adult Endocrinology, NICHD, Bethesda, MD

For further information, contact rennerto@mail.nih.gov or visit http://scg.nichd.nih.gov.