
- Identification of copy number variants associated with renal agenesis using array-based comparative genomic hybridization Chen, Beichen https://iro.uiowa.edu/discovery/delivery/01IOWA_INST:ResearchRepository/12730670610002771?l#13730820860002771 Chen, B. (2010). Identification of copy number variants associated with renal agenesis using array-based comparative genomic hybridization [University of Iowa]. https://doi.org/10.17077/etd.noml5jhf https://iro.uiowa.edu Copyright 2010 Beichen Chen Downloaded on 2021/10/06 12:15:40 -0500 - IDENTIFICATION OF COPY NUMBER VARIANTS ASSOCIATED WITH RENAL AGENESIS USING ARRAY-BASED COMPARATIVE GENOMIC HYBRIDIZATION by Beichen Chen A thesis submitted in partial fulfillment of the requirements for the Master of Science degree in Biology in the Graduate College of The University of Iowa July 2010 Thesis Supervisor: Assistant Professor John R. Manak Graduate College The University of Iowa Iowa City, Iowa CERTIFICATE OF APPROVAL ___________________________________ MASTER’S THESIS ________________ This is to certify that the Master’s thesis of Beichen Chen has been approved by the Examining Committee for the thesis requirement for the Master of Science degree in Biology at the July 2010 graduation. Thesis Committee: __________________________________ John Manak, Thesis Supervisor __________________________________ Sarit Smolikove __________________________________ Polly Ferguson ACKNOWLEDGMENTS I would like to thank all members in Dr. Manak’s lab, especially to Steven Butcher, Xiaojing Hong and Riley Boland who help me for numerous times in my experiments. Also, thanks to Dr. Brophy and Jason Clarke who provide patients’ DNA samples and useful information for my projects. Thanks to Song Yi and Nidhi Sahni for their nice help in thesis preparation and writing. Thanks to my advisor, Dr. Manak, for his insightful instructions and profound experience in research during my 2-year study in the lab. And in the end, to my parents who always support me under any circumstances. For the love and help from all of you. ii ABSTRACT Copy Number Variants (CNVs) are defined as DNA segments of 1kb or more in length and present in a variable number of copies in the human genome. It has been recently shown that many human genetic diseases including organ malformations are caused by CNVs in a patient’s genome. However, the genetic and molecular basis for Renal Agenesis (RA), which is a medical condition whereby unilateral or bilateral fetal kidneys fail to develop, has not yet been extended to CNV studies. By using array-based Comparative Genomic Hybridization, we are analyzing DNA from patients who have RA in order to identify CNVs that are causative for RA; genes within the CNVs will then be assessed for their potential involvement in RA by altering their dose in Xenopus embryos. iii TABLE OF CONTENTS LIST OF TABLES……………………………………………...………………………... v LIST OF FIGURES………………………………………………………………………vi INTRODUCTION………………………….……………...……………………………...1 MATERIALS AND METHODS………………………………………………………….6 Genomic DNA preparation………………………………………………………..6 DNA labeling………………….…….………………………………………….....6 Array-based CGH………………………..………………………………...……...7 NimbleScan 2.5 Analysis..……………….………………………………...……...7 RESULTS AND DISCUSSION…………………………………………...……………...8 CONCLUSION AND FUTURE STUDIES……………………………………………..44 REFERENCES………………………………………….……………………………….45 iv LIST OF TABLES Table 1. Novel amplifications and deletions detected in RA patients ....................................12 2. 27 CNVs which contain genes or part of gene .........................................................13 3. Genes and their functions contained within the 27 novel CNVs..............................14 4. RA Candidate Genes.................................................................................................26 5. Novel CNVs that do not cover genes and their regulatory elements, ETSs andConservedregions................................................................................................30 6. Novel CNVs with regulatory elements or adjacent genes that are involvedin renal diseases ............................................................................................................34 7. Genes involved in kidney development....................................................................35 8. Race and origin of members who share similar deletion on chromosome 13 ..........39 v LIST OF FIGURES Figure 1. Copy Number Variants. ..............................................................................................4 2. Non-allelic homologous recombination can generate duplications and deletions......................................................................................................................5 3. 2.1M segMNT plot of an amplification on chromosome 14 51,005,999- 54,929,999 in patient JCA27II.1A............................................................................27 4. SignalMap Analysis of the amplification region in Patient JCA27II.1A.. ...............28 5. Regulatory elements, ESTs, Conserved region and repeated sequences within chr13: 57,054,000-57,090,000..................................................................................40 6. Regulatory elements, ESTs, Conserved region and repeated sequences within chr5: 109,301,999-109,385,999................................................................................42 vi 1 INTRODUCTION Renal agenesis (RA) is a congenital kidney malformation disease in which one (unilateral RA) or both (bilateral RA) kidneys fail to develop in a fetus. In children, RA is one of the leading causes of end stage renal disease (ESRD), which alone accounts for $15 billion of annual health care expenditure in the U.S. (USRDS, 1999). Occurrence of Unilateral Renal Agenesis (URA) is 1/1000, while Bilateral Renal Agenesis (BRA) occurs at a frequency of 1/3000-1/5000 (Norwood and Chevalier, 2003; Yalavarthy and Parikh, 2003); the latter is almost always fatal at birth because of the presence of the oligohydramnios sequence in which lack of amniotic fluid results in fatal problems for the fetus (Potter, 1965; Potter 1946). Combining our pedigree analysis (Clarke and Brophy, unpublished data) with current literature, BRA appears to have both autosomal dominant and autosomal recessive hereditary forms (Simone, 2007). Moreover, more than 70 different clinical conditions exist where RA has been identified as a component (Sanna-Cherchi et al., 2007; Kerecuk et al., 2008), and phenotypes of RA, particularly URA, are widely heterogeneous (Yalavarthy and Parikh, 2003). Despite the importance and complexity of RA, researchers still lack an understanding of the precise genetic process leading to RA. There are some genes that have been shown to be associated with RA or pediatric congenital anomalies of the kidney and urinary tract (CAKUT), which is a larger category of renal diseases including RA. These genes, including PAX2, RET, EYA1, PBX1, SIX2, are either derived from animal studies or have been found mutated in human renal syndrome patients (Torres et al., 1995, Schuchardt et al., 1994, Johnson et al.,1999, Schnabel et al., 2003, Simone, 2007). However, due to its complexity, other genes are likely to play a role in RA. Furthermore, human RA has been characterized as a multifactorial disorder with an apparent genetic contribution (Yalavarthy and Parikh, 2003). By understanding its genetic basis, medications and therapy for RA could be improved. Copy number variants (CNVs) are defined as abnormal DNA segments of 1kb or greater that are present in a variable number of copies in the human genome; they are likely to be one of 2 the major factors which induce RA (Fanciulli et al., 2009) (Figure 1). CNVs could either be inherited or de novo genomic rearrangements such as deletions, amplifications and translocations. These amplifications and deletions are usually caused by Non-Allelic Homologous Recombination (NAHR) in which non-allelic repeat sequences pair with each other and recombination takes place between the repeated sequences (Figure 2). Regardless of origin, genomic regions encompassed by CNVs can contain one to hundreds of genes, as well as other functional elements (Redon, 2006). If CNVs contain genes that have important biological functions, their presence could potentially lead to disease. In the last 5 years, the number of diseases identified as being induced by CNVs include various cancers (Kallioniemi, 2008; Lenz et al., 2008), nervous system diseases (Walsh et al., 2008; Dibbens et al., 2009; Glessner et al., 2009; Mefford et al., 2009a, b), and congenital malformation and birth defects (Greenway et al., 2009; Lu et al., 2008; Osoegawa et al., 2008; Shi et al., 2009). One study predicts that at least 10% of sporadic cases of tetralogy of Fallot, a severe congenital organ malformation, are caused by CNVs (Greenway et al., 2009). However, to date, relevant studies for human RA focused either on single nucleotide polymorphisms, gene mutations or linkage analysis, whereas studies of the role of CNVs in RA have been absent. We are seeking to identify causative CNVs in RA patients in order to ultimately identify the genes within CNVs that are associated with RA. To identify CNVs, we use microarray-based Comparative Genomic Hybridization (aCGH). aCGH has identified CNVs associated with diseases such as immunodeficiency virus (HIV), auto immune disease and a spectrum of neuropsychiatric disorders (Gonzales, 2005; Aitman, 2006; Fanciulli, 2007; Willcocks, 2008) as well as the various diseases/disorders
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