GENETIC AND GENOMIC STUDIES IN NORMAL DEVELOPMENT AND DISEASES
, Head, Section on Clinical Genomics
Alan L.Y. Pang, PhD, Senior Research Fellow
Margarita Raygada, PhD, MS, Staff Genetic Counselor
Cigdem F. Dogulu, MD, PhD, Clinical Fellow
We apply genetic and genomic technologies acquired through basic studies to research on clinical problems. One of our missions is to provide training to physicians in the application of genomic and genetic approaches to the study of human diseases. Using the cutting-edge methylation tiling array technology, we have embarked on a study of the transcription regulation of mArd2, a novel testis-specific gene first cloned in this laboratory. Another gene under active study is Lin 28, which has been shown to regulate developmental timing. Proteomic approaches helped identify RNAs bound to this protein. Gene knockdown experiments are under way to study the function of Lin 28 in determining cell fate. Relevant to our mission is an attempt to devise global approaches to the identification and screening of risk factors for complex disorders. To this end, we have designed two hybridization-based high-throughput methods for the screening of susceptibility risk factors for thrombophilia and age-related macular degeneration. Aside from basic and translational research, we undertake clinical protocols to study patients with genetic and metabolic disorders. The protocol service gives us access to various genetic disorders and provides clinical genetics training to our fellows, a critical component of the Program in Reproductive and Adult Endocrinology.
Transcription Regulation and Functional Studies of Testis-Specific Genes
Epigenetic regulation of testis-specific gene expression
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. We showed that, in vitro, the protein encoded by mArd2 is functionally similar to its homologue mArd1. However, the two homologous genes display widely differing expression patterns in various cell lines and mouse tissues. In particular, the mArd2 gene is expressed in a developmentally regulated manner during spermatogenesis. To elucidate the molecular mechanism of its 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 hypothesized that DNA methylation could play a role in the regulation of mArd2 expression. After treating both NIH/3T3 and GC-2spd(ts) cells with 5-aza-2¢-deoxycytidine, we observed reactivation of mArd2 transcription, which supports the idea that DNA methylation is responsible for regulating mArd2 transcription. By sequencing bisulfite-treated genomic DNA isolated from various adult somatic tissues and germ cells at different stages of spermatogenesis, we compared the methylation status of the two CpG islands spanning the proximal promoter and half of the coding region of mArd2 gene. Our result showed that the proximal promoter of mArd2 was hypermethylated in mouse tissues that do not express mArd2 but hypomethylated in male germ cells that do express the gene. Interestingly, 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. Our findings illustrate that the testis-specific expression of mArd2 is epigenetically regulated. The Ard1-Ard2 expression system could be a paradigm for studying epigenetic regulation of mammalian spermatogenic gene expression.
Examination of the biological role of Lin28 in mitotic male germ cells
Another gene we identified from expression profiling of male germ cells is mLin28. Lin28 is an evolutionarily conserved RNA-binding protein that is implicated in the correct temporal control of cellular development. Given that Lin28 is highly expressed only in mitotic male germ cells in the testis, we hypothesized that it could act as an RNA chaperone that controls the availability of transcripts encoding products important to cell fate decision. To prove our hypothesis, we used the P19 embryonal carcinoma cells as a model. Our preliminary findings indicate that the Lin28 protein can bind to specific sets of RNA transcripts that encode ribosomal proteins, components of protein translation, and mRNA processing machineries, and other proteins. Gene knockdown experiments are now under way to confirm the role of Lin28 in regulating the synthesis of these target proteins and to study their involvement in the decision of cellular fate.
Genomic and Genetic Studies of Heritable Disorders
Application of high-throughput approaches in the study of complex disorders
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. It is, however, an avoidable disease if currently available prophylactic treatment is instituted. Our calculations demonstrated that concurrent use of a panel of 11 genetic tests increased by at least 30-fold the positive predictive value of testing for venous thrombosis. 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 145 venous thrombosis–associated recurrent mutations and polymorphisms in nine molecules (antithrombin III [AT III], protein C, protein S, fibrinogen, factor V [FV], prothrombin [factor II], methylenetetrahydrofolate reductase [MTHFR], angiotensin 1-converting enzyme [ACE], and plasminogen activator inhibitor-1 [PAI-1] genes). MERT will help us develop stratification protocols for risk-adapted prophylaxis. We designed 291 oligonucleotide 25-mer probes to be spotted onto the microarray, which permitted us to amplify 40 amplicons covering the variation sequences from nine different genes in a single amplification reaction by Multiplex PCR assay. We are now in the process of verifying our method’s analytic validity.
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], patent pending). 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 by their low predictive value and low ability to detect mutations prevalent only in Caucasian populations. We have designed a MERT-ARMD that, by using hybridization-based, high-density oligonucleotide array technology, will concurrently screen 105 known ARMD–associated mutations and polymorphisms in 16 molecules (CFH, LOC387715, BF, C2, ABCR, Fibulin 5, VMD2, TLR4, CX3CR1, CST3, MnSOD, MEHE, paraoxonase, APOE, ELOVL4, and hemicentin-1 genes).
Identification of the role of susceptibility to thrombosis in pseudotumor cerebri of nephropathic cystinosis
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 (1) nephropathic cystinosis patients who develop PTC and (2) 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 proteins C and S, antithrombin III, fibrinogen, total homocysteine, and 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, the FV G1628A polymorphism, the FV R2 allele, the prothrombin 20210 mutation, and the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene C677T polymorphism. 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) and IgM antibodies in one patient, and activated protein C resistance (APCR) in one patient. TT 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). APCR 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 pediatric and adults patients with genetic and metabolic disorders
We are conducting two active protocols in our program. Protocol #02-CH-0023 aims to provide care for patients with a variety of rare genetic disorders, supplementing and offering additional opportunities for physician training in clinical genetics, dysmorphology, and metabolic genetics, in NICHD and other Institutes of the NIH and stimulating clinical research initiatives. The overall purpose of the protocol is to support the Institute’s training and research missions by expanding the spectrum of diseases that can be seen in our clinics and wards. We study disorders such as 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 a treatment), patients with genetic/metabolic-related conditions may be evaluated under Protocol #02-CH-0023. Under the protocol, we have evaluated a total of 386 patients. In addition, we offer genetic counseling services to patients and their families to assess risk and provide information on preventive measures and testing options. Standard, medically indicated laboratory or radiological studies may be performed to confirm a diagnosis or to aid in the management of the patient. In some cases, the patient receives medical or surgical treatment for his or her 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 and abdominal fat) to breast cancer risk. The ultimate goal is to develop new strategies for risk reduction and early screening of at-risk patients. Our study plan calls for (1) determining whether factors associated with or governing insulin function may be involved in modification of breast cancer risk in specific patient populations; (2) determining the role of abdominal fat and other anthropometric measures in breast cancer risk in patients with and without a positive family history (classified by menopausal status) and the interaction with insulin and other hormones; (3) providing sufficient data for future studies looking at mediators of the actions of insulin on breast cancer (IRS-1 and IRA isoform) and gene polymorphisms involved in this at-risk 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 August 2007, we had recruited 19 patients. Recruitment has slowed due to the decision by the NIH Clinical Center to stop payment for genetic testing. We are in the process of reassessing data analysis and protocol direction.
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;92:779-86.
Publications Related to Other Work
Chan WY, Lee TL, Wu SM, Ruszczyk L, Alba D, Baxendale V, Rennert OM. Transcriptome analyses of male germ cells with serial analysis of gene expression (SAGE). Mol Cell Endocrinol 2006;250:8-19.
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;201-20.
Chan WY, Wu SM, Ruszczyk L, Law E, Lee TL, Baxendale V, Rennert OM. The complexity of antisense transcription revealed by the study of developing male germ cells. Genomics 2006;87:681-92.
Lee TL, Alba D, Wu SM, Baxendale V, Rennert OM, Chan WY. Application of transcriptional network analyses in mouse germ-cell transcriptomes. Genomics 2006;88:18-33.
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 2007 [E-pub ahead of print].
Pang ALY, Johnson W, Dym M, Rennert OM, Chan WY. Expression profiling of purified male germ cells: stage specific expression patterns related to meiosis and post-meiotic development. Physiol Genomics 2006;24:75-85.
Vong QP, Li YM, Lau CYF, Dym M, Rennert OM, Chan WY. Structural characterization and expression studies of Dby and its homologs in the mouse. J Androl 2006;27:653-61.
COLLABORATORS
Wai-Yee Chan, PhD, Program in Reproductive and Adult Endocrinology, NICHD, Bethesda, MD
William Gahl, MD, PhD, Clinical Director, NHGRI, Bethesda, MD
Muriel I. Kaiser, MD, Ophthalmic Genetics and Visual Function Branch, NEI, Bethesda, MD
Stephen Kaler, MD, MPH, Program in Molecular Medicine, NICHD, Bethesda, MD
Chris Y.F. Lau, PhD, University of California San Francisco, San Francisco, CA
Stephanie O. Peacock, BS, Program in Reproductive and Adult Endocrinology, NICHD, Bethesda, MD
Constantine Stratakis, MD, DSc, Program in Developmental Endocrinology and Genetics, NICHD, Bethesda, MD
Yan Su, MD, PhD, Loyola University, Chicago, IL
For further information, contact rennerto@mail.nih.gov.
