Identification of Copy Number Variants Associated with Renal Agenesis Using Array-Based Comparative Genomic Hybridization

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

IDENTIFICATION OF COPY NUMBER VARIANTS ASSOCIATED WITH RENAL AGENESIS USING ARRAY-BASED COMPARATIVE GENOMIC HYBRIDIZATION

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.

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.

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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

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

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

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

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 mentioned above. Compared to traditional cytology technologies such as karyotyping and fluorescence in- situ hybridization whose low resolution cannot detect small deletions in the genome, aCGH can detect CNVs as small as a few kilobases and reveal the fold change quantitatively. Moreover, aCGH can identify disease-causing CNVs in large numbers of patients’ samples at a faster rate than other technologies such as large-scale DNA sequencing. By using aCGH, we wish to identify CNVs which could potentially induce the disease in

RA patients. This first study is limited in BRA patients, since they most likely share a similar disease process which could be result from similar micro-deletions and amplifications in genome. We will also examine patients with URA to determine if the same genetic pathways play a similar role as in BRA. Once we identify CNVs which affect functional genes or regulatory elements, further functional analysis with early Xenopus embryos will be conducted. Genes or genes whose regulatory elements are located within causative CNVs will either be knocked down or overexpressed in the embryo of Xenopus to determine if they function in kidney development.

Figure1. Copy Number Variants.

Copy number variants could be caused by amplification, deletion and translocation. A. Normal copy number. Each of the homologous chromosomes have one copy (green) sided by flanking region (blue). B. Deletions can take place in one or both of the homologous chromosomes and reduce the copy number. C. Amplification can take place in one or both of the homologous chromosomes and increase the copy number. D. Multiple amplifications and different combinations could further alter the copy number.

Figure 2. Non-Allelic Homologous Recombination can generate duplications and deletions.

Green arrows represent repeat sequence on chromosomes. When non-allelic repeat sequences pair with each other and recombination takes place, a duplication is generated on one chromosome and a deletion is generated on the other chromosome.

MATERIALS AND METHODS

Genomic DNA preparation

Blood samples of RA patients were obtained worldwide and provided by Dr. Patrick Brophy’s Laboratory at the University of Iowa Carver College of Medicine. The patient population is mainly Caucusian, with some Hispanics and Middle Easterners as well. Patient IDs labeled with ‘JCA’ were gathered in Ann Harbor, Michigan, and patient IDs which are labeled with ‘JCI’ were gathered in Iowa City, IA. Arabic numbers after ‘JCA’ or ‘JCI’ represents the specific pedigree and roman numbers represent generation in the pedigree. The last Arabic numbers stand for the individual in the generation. Genomic DNA of RA patients and reference samples were extracted from blood. The concentration of DNA samples was between 0.2ug/ul to 1.5ug/ul. Agarose gel electrophoresis was used to ensure there is no degradation in DNA samples.

DNA labeling

1ug of patient DNA was mixed with Cy3-random nonamers and 1ug of reference DNA was mixed with Cy5-random nonamers (NimbleGen Dual-Color DNA Labeling Kit) in a total of 80ul. In short, according to the NimbleGen CGH protocol, both samples were denatured at 98 ºC for 10 min. 2µl of 50U/µl Klenow Fragment (3’->5’ exo-) and 10µl of 10mM dNTP mix were added to each sample after denaturing. 8ul nucleus-free water was added into the PCR tube to bring the total volume to 100ul. The mixed samples were incubated at 37 ºC for 2 hours. 10ul of 0.5M EDTA was added into the tube to stop the reaction. Labeled DNA was mixed with 11.5ul 5M NaCl and the labeled DNA was incubated with 120ul iso-proponal for 10min and centrifuged at 14000rpm for 10min. Supernatant was removed and pellets were rinsed in 80% ice-hold ethanol, then dried down in a Speed-VAC. Dried contents were rehydrated in 50ul of nucleus-free water and the concentration was calculated. 34ug of each labeled DNA was taken out to be dried down in the Speed VAC.

Array-based CGH

The array used in in performing the CGH is a human design 2.1 million feature whole genome tilling array from Roche Nimblegen. (NimbleGen System, Madison, WI). This array covers all chromosomes in the human genome on one array. The length of array probes are 60 bp and the median probe spacing is 1169 bp. 34ug of test sample was rehydrated in hybridization buffer with an equal amount of reference sample and loaded onto the array. The array was incubated on the NimbleGen Hybridization System for 60-72 hour at 42 ºC before unbound fluorescent DNA was washed off. The array was scanned by a GenePix 4400A scanner at a resolution of 2.5um using GenePix 7.0 software.

NimbleScan 2.5 Analysis

The scanned image corresponding to both the Cy3 and Cy5 hybridization was loaded into the NimbleScan 2.5 software and the log 2 ratio of fluorescent intensity of Cy3/Cy5 was calculated. Segment plots were then generated after the images were processed; such plots allow the identification of CNVs due to the fact that Cy3/Cy5 ratios will change when DNA copy numbers are different between the case and control samples. In the algorithm for calculating different segments on a chromosome, 0.1 is minimum difference for two segments to be considered as separate. Each segment is comprised of at least 2 probes. In average segment plots, averaging windows are 10X, which means that each window-averaged data point represents the average of 10 original data points contained within a 10,000 bp window. For unaveraged segment plots, the averaging window is 1X.

RESULTS AND DISCUSSION

We screened patients with BRA either confirmed by ultrasound or autopsy. Patient DNA samples were hybridized onto the Roche Nimblegen 2.1 million feature human whole genome tiling arrays and the resulting data was analyzed for evidence of CNVs. In the segmentation plots (see Figure 3), the X-axis represents genome position while the Y-axis represents the log2 ratio of RA patient (case) DNA over reference DNA. For an amplification, the log2 ratio is above zero, while for a deletion, the log2 ratio is below zero in the segmentation profile. We consider any amplifications whose log 2 ratio is above 0.3 and any deletions whose log 2 ratio is below -0.5 as significant. 20.3=1.23. Ratio of patients’ DNA amount to reference DNA amount is 1.23. 2-0.5=0.71. Ratio of patients’ DNA amount to reference DNA amount is 0.71. For duplications and heterozygous deletions, shift values of 0.4 and -0.7, respectively, are usually obtained. However, we chose 0.3 and -0.5 as conservative parameters in order to prevent us from missing CNVs in our analysis. CNVs can be one of two types: common CNVs (also called Copy Number Polymorphisms) present in healthy individuals, or rare CNVs which can be specific to a disease such as BRA. For all CNVs we have identified, we compared them to the common CNVs in the Database of Genomic Variants (DGV; http://projects.tcag.ca/variation/) which contains a large repository of

CNVs shared by healthy individuals in order to highlight the CNVs that are potentially unique and causative to BRA. We discovered 1371 amplifications and deletions in total. 855 of them are amplifications and 518 are deletions (Table 1). By comparing these 1371 CNVs to DGV, we found 57 amplifications and deletions which are not covered by any common CNVs and that have ever been detected in healthy individuals. These 57 novel amplifications and deletions could be classified into two categories. The first category includes CNVs that contain genes or parts of genes (Table 2). This category has 27 CNVs. Genes that they cover and functions of these genes

are listed in Table 3. The second category includes CNVs that do not overlap with any known genes and has 30 CNVs in total (Table 5). When analyzing the first category, we used SignalMap 1.9.0.05 to visualize the primary transcripts of all known genes and their transcription starting sites within the novel CNVs. There are 55 genes altogether, and the protein products of 43 primary transcripts have been characterized in some detail while the other 12 gene products remain less well characterized. The functions of these genes are highly diverse and involved in various aspects of biological processes including signal transduction, cell cycle regulation, enzyme processes and cell adhesion. Importantly, 7 genes have been shown to be expressed in kidney: BMP4, CNIH, NID2, SPINK5, PTPRK, RHPN2 and CRLF2. Considering RA published research in the NCBI database and a candidate gene list of RA which was derived either from animal studies or some human genetic syndromes (Table 4), bone morphogenetic protein 4 (BMP4) is one of the genes that are worthy of notice within the amplification region on chromosome 14 of case JCA27II.1A (Figure 3). Patient JCA27II.1A has a unique amplification on chromosome 14: 51,005,999-54,929,999 (Figure 3); although several small CNVs within this region have been identified in controls (Database of Genomic Variants), no large deletions encompassing this amplification have been identified. This indicates that the amplification region could potentially be a CNV that is causative for BRA rather than a common CNV.

The size of whole amplification region on chromosome 14 is 3,924,000bp, the largest one in the pool of our novel CNVs. The log 2 ratio of the fluorescence intensity of the patient’s sample to reference samples was determined by the segmentation algorithm to be 0.316. However, considering the average level of log 2 ratio of the chromosome near the amplification, which is 0.1 lower than zero line, the actual log2 ratio of this amplification is 0.416, and accordingly, there is 1.33 times more of this DNA region on chromosome 14 in the case. BMP4, a member of the TGF-β superfamily, has been shown to be involved in many aspects of embryonic development ranging from establishing dorsal-ventral axis, bone and

cartilage development, tooth and limb development, fracture repair, muscle development and bone mineralization, and more importantly to this study, uteric bud development. BMP4, expressed in lung and, at a lower level in kidneys, also takes part in morphogenesis of many organs, especially originated from mesoderm as BMP4 is crucial for mesoderm formation (Hogan BL, 1996; Ducy P, Karsenty G, 2000). In 2000, Miyazaki et al. showed that BMP4 has two functions in ureteric bud development. On the one hand, BMP4 inhibits the ectopic budding from the Wolffian duct (WD) and ureter stalk. On the other hand, in the later stage of nephrogenesis, BMP4 has the ability to promote the elongation of the branching ureter within the metanephros and further promotes kidney morphogenesis (Miyazaki et al., 2000). In their study, they placed BMP4-loaded beads adjacent to the second branching point of E11.5 mouse metanephric explants where Bmp4 is not expressed under normal condition. The branching of ureteric bud was significantly inhibited in the surrounding area compared to the area where there is no BMP4-loaded bead. Later on they checked the expression of Wnt11 which is a molecular marker for ureteric buds, and found expression of Wnt11 was downregulated in the adjacent branching ureter after 8 hours incubation. Moreover, BMP4 has been identified in pediatric congenital anomalies of kidney and urinary tract (CAKUT) which represents a major cause of chronic renal failure that includes RA (Tabatabaeifar et al., 2009). By studying the cellular expression pattern of BMP4 mutant patients, Tabatabaeifar concluded that mutations in BMP4 are responsible for abnormalities of human kidney development in CAKUT. Another 6 genes within the amplification are involved in cell adhesion (such as NID2), singal transduction (such as CRLF2), and other processes, although there is no literature in NCBI showing that they are related to renal diseases up to now. Nonetheless, some of these genes are expressed in kidneys at different levels. This means they could potentially affect renal development. As a result, functional analysis of these genes will have priority in our future work. The second category contains novel CNVs that do not cover any known genes; however,

these CNVs could either contain regulatory elements of adjacent genes or have EST/mRNA transcripts which could represent new genes, or novel exons of known genes, within them, and we have identified several of these transcripts in the CNV intervals (Table 5). Also, all but 2 of them have highly conserved regions through vertebrate evolution (Table 5) as determined by conservation plots in the UCSC genome browser. As a result, they are still potentially involved in RA. It is clear that some of nearby genes of these CNVs has been shown to be associated with renal diseases, such as EYA1, which has been shown to be involved in branchio-oto-renal (BOR) syndrome (Johnson, 1999). It could be possible that the novel CNVs affect regulatory elements of the nearby genes. In order to test this hypothesis, we screened these novel CNVs for any putative regulatory elements potentially regulated by transcription factors expressed in the kidney and/or associated with kidney disease using the USCS human genome browser (NCBI36/hg18; see Table 5 and Figure 5 for examples). Considering that the list of RA candidate genes includes genes encoding transcription factors, several of the intergenic, novel CNVs we identified contain conserved binding sites that are worthy of discussion. (Table 6). PAX2, EYA1, PBX1, HOXA9, LHX3, OCT1 have all been implicated in renal diseases by previous studies (Torres et al., 1995; Johnson et al., 1999; Schnabel et al., 2003; Pavlova A et al., 2000; Akio Kobayashi et al., 2004; Davis AP et al., 1995; Shen-Ju Chou, 2006). The function of each of these genes is described in Table 7. Intriguingly, conserved binding sites for all of these factors can be found in several of the novel CNVs we identified (Table 6).

Table 1. Amplifications and deletions detected in RA patients.

Patient Amplificaton Deletion JCA4II.2B 24 31 JCA4II.3C 31 34 JCA14II.1F 31 2 JCA34II.1C 32 3 JCA45II.1A 39 6 JCI35II.2A 17 9 JCA30II.1B 9 6 JCA11III.1A 27 24 JCA14II.1D 37 17 JCA29III.6B 11 12 JCA29III.1A 10 7 JCA32II.1D 28 12 JCI39II.2A 24 19 JCA35II.1A 12 16 JCA18III.7K 26 13 JCA27II.1A 23 26 JCA29III.5B 25 19 JCA23II.4A 22 11 JCA18III.4A 19 9 JCA18III.6M 12 15 JCA16II.2A 11 8 JCA18II.4B 10 4 JCA22II.1A 28 14 JCA5II.1A 12 13 JCA11II.1A 13 12 JCA15.2C 30 15 JCA18III.3B 20 30 JCA28II.2E 60 13 JCI4611.1A 45 18 JCA15II.3E 62 28 JCA31II.1A 42 13 JCA18II.6A 10 11 JCA45II.1B 37 37 JCI44II.1A 16 11

Table 2. 27 Novel CNVs which affect gene structure.

Patient Chr Start Point Stop Point Size of Ratio Genes within the CNVs region JCA18III.7K chr1 28349999 28385999 36000 0.3281 PTAFR JCA27II.1A chr2 1.71E+08 1.71E+08 24000 0.4874 MYO3B, SP5 JCA27II.1A chr2 70025999 70097999 72000 0.3054 MXD1, ASPRV1 JCA14II.1D chr2 1.97E+08 1.97E+08 12000 0.5164 DNAH7 JCA11II2A chr5 7470000 7518000 48000 0.4248 ADCY2 JCA18II6A chr5 59754000 59802000 48000 0.3676 PART1 JCA23II4A chr5 1.47E+08 1.47E+08 120000 ‐0.6530 SPINK5 JCI35II2A chr5 93701999 93761999 60000 ‐0.5134 C5 or f36 JCA11III1A chr6 1.29E+08 1.29E+08 372000 0.4776 PTPRK JCA29III.6B chr7 1.58E+08 1.58E+08 72000 ‐0.5420 NCAPG2 JCA29III.6B chr7 1.58E+08 1.58E+08 228000 0.4258 PTPRN2 JCA11II2A chr8 72786000 72894000 108000 ‐0.8400 MSC JCA27II.1A chr10 99497999 99533999 36000 0.3050 ZFYVE27,SFRP5 JCI39II.2A chr10 73673999 73793999 120000 0.3062 DDIT4 DNAJB12 JCA29III.5B chr11 43805999 43913999 108000 0.4356 ALKBH3 JCA29III.6B chr11 43817999 43985999 168000 0.4343 ALKBH3 JCA35II.1A chr12 98705999 98765999 60000 ‐0.7464 ANKS1B JCA29III.5B chr13 19325999 19337999 12000 0.4115 ZMYM5 JCA27II.1A chr14 51005999 54929999 3924000 0.3164 BMP4, CDKN3, GCH1.. JCA29III.1A chr14 23981999 24065999 84000 0.3151 CMA1 JCI39II.2A chr16 67253999 67421999 168000 0.4498 CDH1, CHD3 JCA35II.1A chr18 17045999 17225999 180000 ‐0.716 KIAA1772 JCA15II2C chr19 38178000 38214000 36000 0.3125 RHPN JCA27II.1A chr19 38177999 38201999 24000 0.3518 RHPN2 JCA34II.1 chrX 1.53E+08 1.53E+08 96000 0.4285 OPN1MW,TEX28,TKTL1 JCA18III6M chrX 1134000 1290000 156000 0.3706 CRLF2

Table 3. Genes and their functions contained within the 27 novel CNVs.

NCBI Gene Protein Products Family Function Tissue Speficity ID Name 652 BMP4 Bone Morphogenic Bone Early diffrentiation of embryo; Expressed in the lung and Proteins 4 Morphogenic establishing dorsal‐ventral axis; lower levels seen in the Proteins involves in bone and cartilage kidney. Present also in normal development; tooth and limb and neoplastic prostate development; fracture repair; tissues, and prostate cancer muscle development; bone cell lines. mineralization; uteric bud development 1033 CDKN3 Cyclin‐dependent kinase Dual specificity May play a role in cell cycle inhibitor 3 protein regulation. Dual specificity phosphatase phosphatase active toward family substrates containing either phosphotyrosine or phosphoserine residues. Dephosphorylates CDK2 at 'Thr‐160' in a cyclin‐dependent manner. 2643 GCH1 GTP cyclohydrolase I GTP Positively regulates nitric oxide In epidermis, expressed (GTPCH) cyclohydrolase synthesis in umbilical vein predominantly in basal family endothelial cells (HUVECs). May be undifferentiated keratinocytes involved in dopamine synthesis. and in some but not all May modify pain sensitivity and melanocytes (at protein level). persistence. Isoform GCH‐1 is the functional enzyme, the potential function of the enzymatically inactive isoforms remains unknown. 1 4

Table 3. continued 2764 GMFB Glial maturation Factor‐ Glial maturation Phenotypic differentiation of glial Largely limited to the brain beta Factors cells and neruons 3958 LGALS3 Galectin‐3 Lectin Galactose‐specific lectin which A major expression is found in binds IgE. May mediate with the the colonic epithelium. It is alpha‐3, beta‐1 integrin the also abundant in the activated stimulation by CSPG4 of macrophages. endothelial cells migration. Together with DMBT1, required for terminal differentiation of columnar epithelial cells during early embryogenesis 5706 PSMC6 26S protease regulatory Triple‐A family of The 26S protease is involved in the Cytoplasm and Nucleus subunit S10B ATPases ATP‐dependent degradation of ubiquitinated proteins. The regulatory (or ATPase) complex confers ATP dependency and substrate specificity to the 26S complex.

5729 PTGDR Prostaglandin D2 receptor G‐protein coupled Receptor for prostaglandin D2 Expressed in retinal choroid, (DP1) receptor (PGD2). The activity of this ciliary epithelium, longitudinal receptor is mainly mediated by and circular ciliary muscles, G(s) proteins that stimulate iris, small intestine and adenylate cyclase, resulting in an platelet membranes. elevation of intracellular cAMP. A mobilization of calcium is also observed, but without formation of inositol 1,4,5‐trisphosphate 1 5

Table 3. continued 5732 PTGER2 Prostaglandin E2 receptor G‐protein coupled Receptor for prostaglandin E2 Placenta and lung. EP2 subtype receptor (PGE2). The activity of this receptor is mediated by G(s) proteins that stimulate adenylate cyclase. The subsequent raise in intracellular cAMP is responsible for the relaxing effect of this receptor on smooth muscle. 6815 STYX Serine/threonine/tyrosine Probable pseudophosphatase. interacting protein Contains a Gly residue instead of a conserved Cys residue in the dsPTPase catalytic loop which renders it catalytically inactive as a phosphatase. The binding pocket is however sufficiently preserved to bind phosphorylated substrates, and maybe protect them from phosphatases. Seems to play a role in spermiogenesis 9787 DLG7 Disks large‐associated Kinetochore Potential cell cycle regulator that Abundantly expressed in fetal protein 5 Protein may play a role in carcinogenesis of liver. Expressed at lower levels cancer cells. Mitotic in bone marrow, testis, colon, phosphoprotein regulated by the and placenta. ubiquitin‐proteasome pathway. Key regulator of adherens junction integrity and differentiation that may be involved in CDH1‐mediated adhesion and signaling in epithelial cells. 1 6

Table 3. continued 10175 CNIH Protein cornichon homolog Cornichon family Involved in the selective transport Highly expressed in heart, and maturation of TGF‐alpha liver, skeletal muscle, family proteins. pancreas, adrenal medulla and cortex, thyroid, testis, spleen, appendix, peripheral blood lymphocytes and bone marrow. Lower expression found in brain, placenta, lung, kidney, ovary, small intestine, stomach, lymph node, thymus and fetal liver. 10688 CGRRF1 Cell growth regulator with Able to inhibit growth in several ring finger domain 1 cell lines 10979 PLEKHC1 Fermitin family homolog 2 Kindlin family Participates in the connection Ubiquitous. Found in between ECM adhesion sites and numerous tumor tissues. the actin cytoskeleton and also in the orchestration of actin assembly and cell shape modulation. Recruits migfilin (FBLP1) protein to cell‐ECM focal adhesion sites. 11169 WDHD1 WD repeat and HMG‐box Acts as a replication initiation DNA‐binding protein 1 factor that brings together the MCM2‐7 helicase and the DNA polymerase alpha/primase complex in order to initiate DNA replication. 1 7

Table 3. continued 22795 NID2 Nidogen‐2 Cell adhesion glycoprotein which is Heart, placenta and bone. widely distributed in basement Less in pancreas, kidney membranes; Binds to collagens I and skeletal muscle. and IV, to perlecan and to laminin 1; probably has a role in cell‐ extracellular matrix interactions 22863 KIAA0831 23034 SAMD4A Sterile alpha motif domain containing 4A 30001 ERO1L ERO1‐like protein alpha EROs family Essential oxidoreductase that Widely expressed at low level. oxidizes proteins in the Expressed at high level in endoplasmic reticulum to produce upper digestive tract. Highly disulfide bonds. Its reoxidation expressed in esophagus. probably involves electron transfer Weakly expressed in stomach to molecular oxygen via FAD. Acts and duodenum. independently of glutathione. May be responsible for a significant proportion of reactive oxygen species (ROS) in the cell, thereby being a source of oxidative stress. Required for the folding of immunoglobulin proteins. 51637 C14 or f166 54331 GNG2 Guanine nucleotide‐ Guanine A modulator or transducer in Expressed in fetal tissues, binding protein subunit nucleotide binding various transmembrane signaling including testis, adrenal gland, gamma‐2 (G) proteins systems. The beta and gamma brain, white blood cells and chains are required for the GTPase brain. activity, for replacement of GDP by GTP, and for G protein‐effector

interaction 1 8

Table 3. continued 55030 FBXO34 F‐box protein 34 Substrate‐recognition component of the SCF (SKP1‐CUL1‐F‐box protein)‐type E3 ubiquitin ligase complex 57544 KIAA1344 Thioredoxin domain‐ containing protein 16 64841 GNPNAT1 Glucosamine 6‐phosphate Acetyltransferase Catalysis: Acetyl‐CoA + D‐ N‐acetyltransferase family glucosamine 6‐phosphate = CoA + N‐acetyl‐D‐glucosamine 6‐ phosphate. 5724 PTAFR Platelet‐activating factor G‐protein coupled Receptor for platelet activating Expressed in the placenta, receptor receptor 1 family. factor, a chemotactic phospholipid lung, left and right heart mediator that possesses potent ventricles, heart atrium, inflammatory, smooth‐muscle leukocytes and differentiated contractile and hypotensive HL‐60 granulocytes. activity. Seems to mediate its action via a G protein that activates a phosphatidylinositol‐ calcium second messenger system. 4084 MXD1 MAX dimerization protein 1 MAX‐interacting MAX dimerization protein belongs proteins to a subfamily of MAX‐interacting proteins. This protein competes with MYC for binding to MAX to form a sequence‐specific DNA‐ binding complex, acts as a transcriptional repressor (while MYC appears to function as an activator) and is a candidate tumor suppressor. 19

Table 3. continued 151516 ASPRV1 Retroviral‐like aspartic Expressed primarily in the protease 1 granular layer of the epidermis and inner root sheath of hair follicles. In psoriatic skin, expressed throughout the stratum corneum. In ulcerated skin, expressed in the stratum granulosum of intact epidermis but almost absent from ulcerated regions. Expressed in differentiated areas of squamous cell carcinomas but not in undifferentiated tumors. 56171 DNAH7 Dynein heavy chain 7 dynein heavy Force generating protein of Detected in brain, testis and chain family. respiratory cilia. Produces force trachea. towards the minus ends of microtubules. Dynein has ATPase activity; the force‐producing power stroke is thought to occur on release of ADP 108 ADCY2 Adenylate cyclase type 2 adenylyl cyclase A membrane‐bound, calmodulin‐ Expressed in brain. class‐4/guanylyl insensitive adenylyl cyclase. cyclase family. 2 0

Table 3. continued

11005 SPINK5 Serine protease inhibitor Serine protease inhibitor, probably Highly expressed in the Kazal‐type 5 important for the anti‐ thymus. Also found in the oral inflammatory and/or antimicrobial mucosa, parathyroid gland, protection of mucous epithelia. Bartholin's glands, tonsils, and vaginal epithelium. Very low levels are detected in lung, kidney, and prostate. 5796 PTPRK Receptor‐type tyrosine‐ Regulation of processes involving High levels in lung, brain and protein phosphatase kappa cell contact and adhesion such as colon; less in liver, pancreas, growth control, tumor invasion, stomach, kidney, placenta and and metastasis. Forms complexes mammary carcinoma. with beta‐catenin and gamma‐ catenin/plakoglobin. Beta‐catenin may be a substrate for the catalytic activity of PTP‐kappa. 54892 NCAPG2 Condensin‐2 complex Regulatory subunit of the subunit G2 condensin‐2 complex, a complex which establishes mitotic chromosome architecture and is involved in physical rigidity of the chromatid axis. 5799 PTPRN2 Receptor‐type tyrosine‐ protein‐tyrosine Implicated in development of Highest levels in brain and protein phosphatase N2 phosphatase nervous system and pancreatic pancreas. Lower levels in family endocrine cells. trachea, prostate, stomach and spinal chord. 21

Table 3. continued

118813 ZFYVE27 Zinc finger FYVE domain‐ Defects in ZFYVE27 are the cause containing protein 27 of spastic paraplegia autosomal dominant type 33 (SPG33). Spastic paraplegia is a neurodegenerative disorder characterized by a slow, gradual, progressive weakness and spasticity of the lower limbs. Rate of progression and the severity of symptoms are quite variable. 6425 SFRP5 Secreted frizzled‐related Soluble frizzled‐related proteins Highly expressed in the retinal protein 5 (sFRPS) function as modulators of pigment epithelium (RPE) and Wnt signaling through direct pancreas. Weak expression in interaction with Wnts. They have a heart, liver and muscle. role in regulating cell growth and differentiation in specific cell types. SFRP5 may be involved in determining the polarity of photoreceptor, and perhaps, other cells in the retina.

54541 DDIT4 DNA damage‐inducible Inhibits cell growth by regulating Broadly expressed, with transcript 4 protein the FRAP1 pathway upstream of lowest levels in brain, skeletal the TSC1‐TSC2 complex and muscle and intestine. Up‐ downstream of AKT1. Promotes regulated in substantia nigra neuronal cell death. neurons from Parkinson disease patients (at protein level). 54788 DNAJB12 22

Table 3. continued

221120 ALKBH3 Alpha‐ketoglutarate‐ Dioxygenase that repairs alkylated Ubiquitous. Detected in heart, dependent dioxygenase DNA containing 1‐methyladenine pancreas, skeletal muscle, alkB homolog 3 and 3‐methylcytosine by oxidative thymus, testis, ovary, spleen, demethylation. Has a strong prostate, small intestine, preference for single‐stranded peripheral blood leukocytes, DNA. May also act on RNA. urinary bladder and colon. Requires molecular oxygen, alpha‐ ketoglutarate and iron.

27253 PCDH17 Protocadherin‐17 Potential calcium‐dependent cell‐ adhesion protein. 85415 RHPN2 Rhophilin‐2 RHPN family Binds specifically to GTP‐Rho. May Widely expressed. Highly function in a Rho pathway to limit expressed in prostate, stress fiber formation and/or trachea, stomach, colon, increase the turnover of F‐actin thyroid and pancreas. structures in the absence of high Expressed at lower level in levels of RhoA activity. brain, spinal cord, kidney, placenta and liver. 114822 RHPN Rhophilin‐1 RHPN family Has no enzymatic activity. May serve as a target for Rho, and interact with some cytoskeletal component upon Rho binding or relay a Rho signal to other molecules 56899 ANKS1B 9205 ZMYM5 Zinc finger MYM‐type protein 5 1215 CMA1 CMA1 protein peptidase S1 family 2 3

Table 3. continued

999 CDH1 Cadherin‐1 CDH1 is involved in mechanisms Non‐neural epithelial tissues. regulating cell‐cell adhesions, mobility and proliferation of epithelial cells. Has a potent invasive suppressor role. It is a ligand for integrin alpha‐E/beta‐7. 64109 CRLF2 Cytokine receptor‐like type I cytokine Receptor for thymic stromal Expressed in heart, skeletal factor 2 receptor family lymphopoietin (TSLP). Forms a muscle, kidney and adult and functional complex with TSLP and fetal liver. Primarily expressed IL7R which is capable of in dendrites and monocytes. stimulating cell proliferation Weakly expressed in T‐cells. through activation of STAT3 and STAT5. Also activates JAK2. Implicated in the development of the hematopoietic system. 80821 DDHD1 Phospholipase DDHD1 PA‐PLA1 family Probable phospholipase that Highly expressed in testis. Also hydrolyzes phosphatidic acid. The expressed in brain, spleen and different isoforms may change the lung. Only expressed in substrate specificity cerebellum in fetal brain. 93487 C14 or f32 122786 FRMD6 FERM domain‐containing Cytoplasm; Cell membrane; protein 6 Peripheral membrane protein; Cytoplasmic side. Note: Can colocalize with actin 24

Table 3. continued

122809 SOCS4 Suppressor of cytokine SOCS family SOCS family proteins form part of a signaling 4 classical negative feedback system that regulates cytokine signal transduction. May be a substrate recognition component of a SCF‐ like ECS (Elongin BC‐CUL2/5‐SOCS‐ box protein) E3 ubiquitin‐protein ligase complex which mediates the ubiquitination and subsequent proteasomal degradation of target proteins 145438 C14 or f82 283554 GPR137c Integral membrane protein GPR137C 645380 LOC645380 645393 LOC645393 645417 LOC645417 645602 LOC645602

Table 4. RA Candidate Genes. *

Nuclear proteins/Transcription factors Secreted factors/Other Receptors

Pax8 Emx2 Upk3a Bmp4 Ret Hoxa11 Robo2 (Bouchard et al., (Miyamoto et al., (Schönfelder et al., (Miyazaki et al., (Schuchardt et (Davis et al.1995) (Lu et al., 2007) 2002) 1997) 2006) 2000) al., 1994)

Fras1 Six1,4,6 Bmp7 Gfra1 Agtr2 Pax2 Hoxd11 (McGregor et al., (Xu et al., 2003) (Dudley et al., (Enomoto et al., (Nishimura et al (Torres et al., 1995) (Davis et al.1995) 2003; (OMIM 113650) 1995) 1998) 1999) OMIM 219000)

Eya1 Gata3 Wnt4 Six2,3 Prokr2 Rara (Johnson et (Hernández et Spry (Stark et al., (Self et al., 2006) (Dodé et al., (Mendelsohn et al.,1999) al., 2007; OMIM (Rozen et al., 2009) 1994; (OMIM # 113650) 2006) al., 1994) (OMIM 113650) 146255) OMIM # 277000)

Gdnf Sall1 Six5 Pbx1 Wnt11 Rarb (Sanchez et al., Itgb1 (Nishinakamura et (Hoskins et al., (Schnabel et al., (Majumdar et (Mendelsohn et 1996; Skinner et al., (Wu et al 2009) al., 2001) 2007) 2003) al., 2003) al., 1994) 2008)

Crebbp (OMIM # Fgfr1 Sall2 Lhx1 Insl3 Slit2 180849) (Poladia et al., (Shawlot and 2006; (Sato et al. , 2003) Behringer, 1995) (Fu et al., 2005) (Lu et al., 2007) (Bartsch O, 2002; OMIM # 147950) Petrij F, 1995)

Sall3 Gli3 Kal1 Foxd1 Fgfr2 (Böse et al., (Georgopoulos et Slit3 (Hatini et al., (Poladia et al., (Parrish et al., 2002; OMIM al., 2007; OMIM (Liu et al , 2003) 1996) 2006) 2004) 146510) 308700)

Tbx3 Frem2 Kit Sall4 Wt1 (OMIM 181450) Fgf8 (OMIM 219000) (Sakaki‐Yumoto, (Kreidberg et al., (Perantoni et al., (Schmidt‐Ott et 2006) 1993) (Hofstetter et al., 2005) (Jadeja S, 2005) al., 2006) 2008

* Provided by Dr. Patick Brophy.

Many of RA candidate genes including some of components of their pathways are listed. The list of genes has been derived from animal studies and some human genetic syndromes. These genes are involved in various functions during nephrogenesis development ranging from initial induction to tubulogenesis. Some of these genes are also involved in important cellular process including genome stability and DNA repair.

Figure 3. 2.1M segMNT plot of an amplification on chromosome 14 51,005,999- 54,929,999 in patient JCA27II.1A. For the array CGH plots, probes are ordered on the X-axis according to their physically mapped position on chromosomes. The top plot shows the segmentation pattern for a normal chromosome 14: 51,005,999-54,929,999 and adjacent region while the bottom plot shows segmentation pattern for an abnormal chromosome 14: 51,005,999-54,929,999 in patient JCA27II.1A. The maternal grandfather of the patient has a medical condition in which only one kidney exists. The mother of the patient has a history of renal issues although both kidneys exist.

BMP4

Figure 4. SignalMap Analysis of the amplification region in Patient JCA27II.1A. 2 8

(top panel). NimbleGen whole genome tiling array CGH segmentation profile of chromosome 14: 51,005,999-54,929,999 for patient JCA27II.1. (middle panel). Common CNVs on chromosome 14 51,005,999-54,929,999 and adjacent region. Green bars stand for common CNVs that have been detected in healthy individuals in DGV. (lower panel). Purple bars stand for genes within the region.

Table 5. Novel CNVs that do not cover genes and their regulatory elements, ESTs and conserved regions.

Pati Chr Start Point Stop Point Size of Ratio Adjacent Transcription factor binding site ESTs with Conse‐ ent CNVs Genes intron/ESTs rved without intron / Region mRNA s JCA1 chr8 72786000 72894000 108000 ‐0.84008 EYA1 / MSC BRN2, CHX10, FOX1, MEF2,RP58, 4 / 4 / 0 y 1II2 SRF, RFX1, CEBP, NFAT, EVI1, A GCNF, CDP, CART1, PPARG, NKX25, FAC1, HMX1, SEF1, ROAZ JCA1 chr1 57054000 57090000 36000 ‐0.62275 / PCDH17 RP58, OCT1, LHX3, POU6F1, 0 / 6 / 0 Y 1III1 3 MEISIBHOXA9, HNF1, PBX1, E2F, A RORA1, RORA2, NKX61, POU6F1 JCA1 chr1 88721999 88812636 90637 0.48847 PRDM7 POU3F2, NRSF, EVI1, CDP, PBX1, >100 / >100 Y 4II.1 6 GAS8/ AHOXA9, PPARA, PAX2, MRF2, D MZF1, SEF1, PAX4, MIF1, AREB6, CDC5, FOX03 JCA1 chr1 18450000 18486000 36000 1.07976 /DKFZp686a NO 2 / 21 / 4 Y 4II.1 3 1627 F JCA1 chr5 1.09E+08 1.09E+08 84000 0.30888 MAN2A1 / EVI1, FOXD3, ISRE, FOXJ2, OCT1, 0 / 1 / 0 Y 4II.1 FLJ43080 PBX1, NKX3A, CDC5, OCT, LUN1, F CDP, NHF1, TST1, CART1, JCA1 chr1 57042000 57090000 48000 ‐0.64287 / PCDH17 RP58, OCT1, LHX3, POU6F1, 0 / 6 / 0 Y 5II2 3 MEISIBHOXA9, HNF1, PBX1, E2F, C RORA1, RORA2, NKX61, POU6F1 JCA1 chr1 57053999 57089999 36000 ‐0.57056 / PCDH17 RP58, OCT1, LHX3, POU6F1, 0 / 6 / 0 Y 8III. 3 MEISIBHOXA9, HNF1, PBX1, E2F, 7K RORA1, RORA2, NKX61, POU6F1

3 0

Table 5. continued

JCA1 chr1 1.05E+08 1.05E+08 72000 0.30515 AMY1A/ MEF, NKX61, BACH1, BRN2, HNF1, 0 / 2 / 0 Y 8III4 PRMT6 HOXA3, MEF2 A JCA2 chr1 57054000 57090000 36000 ‐0.62577 / PCDH17 RP58, OCT1, LHX3, POU6F1, 0 / 6 / 0 Y 2II1 3 MEISIBHOXA9, HNF1, PBX1, E2F, A RORA1, RORA2, NKX61, POU6F1 JCA2 chrX 56490000 56526000 36000 1.33944 KLF8 / NO NO Y 2II1 UBQLN2 A SPIN3 JCA2 chr1 57054000 57090000 36000 ‐0.6579 / PCDH17 RP58, OCT1, LHX3, POU6F1, 0 / 6 / 0 Y 3II4 3 MEISIBHOXA9, HNF1, PBX1, E2F, A RORA1, RORA2, NKX61, POU6F1 JCA2 chr1 57053999 57089999 36000 ‐0.59645 / PCDH17 RP58, OCT1, LHX3, POU6F1, 0 / 6 / 0 Y 7II.1 3 MEISIBHOXA9, HNF1, PBX1, E2F, A RORA1, RORA2, NKX61, POU6F1 JCA2 chr1 43949999 43961999 12000 0.45981 ALKBH3 / NO 7 / 0 / 0 Y 9III. 1 ASSC EXT2 5B JCA2 chr1 42413999 43985999 12000 ‐0.51105 ALKBH3 / NO 7 / 0 / 0 Y 9III. 1 ASSC EXT2 6B JCA3 chrX 36641999 36713999 72000 0.34864 CX of f30 / PAX4, BACH1 0 / 1 / 0 Y 0II1 FAM47C B JCA3 chr1 57053999 57101999 48000 ‐0.66508 / PCDH17 RP58, OCT1, LHX3, POU6F1, 0 / 6 / 0 Y 2II.1 3 MEISIBHOXA9, HNF1, PBX1, E2F, D RORA1, RORA2, NKX61, POU6F1 JCA3 chr5 1.09E+08 1.09E+08 84000 ‐0.51128 MAN2A1 / EVI1, FOXD3, ISRE, FOXJ2, OCT1, 0 / 1 / 0 Y 2II.1 FLJ43080 PBX1, NKX3A, CDC5, OCT, LUN1, 3

D CDP, NHF1, TST1, CART1, 1

Table 5. continued JCA3 chrX 56381999 56405999 24000 0.35528 KLF8 / NO NO Y 2II.1 UBQLN2 D SPIN3 JCA3 chrX 79193999 79205999 12000 0.30489 TBX22 / NO 1 / 0 / 0 N 2II.1 FAM46D D JCA3 chr1 1.06E+08 1.07E+08 240000 0.54003 / PRMT6 CDPCR, CDP, E4BP4, LHX3, MEIS1, 6 /14/ 0 Y 4II.1 NTNG1 HFH1, MO2COM, AHOXA9 C JCA3 chr1 57222000 57306000 84000 0.55618 PCDH15 / ISER, NKX25, NKX61, FREAC4, 0 /4/0 Y 4II.1 0 ZWINT FOX04, PAX4, EVI1 C JCA3 chr5 12738000 12786000 48000 0.50037 CTNND2 / NO 2+6/ 4 Y 4II.1 DNAH5 C JCA3 chr5 1.09E+08 1.09E+08 84000 0.48523 MAN2A1 / EVI1, FOXD3, ISRE, FOXJ2, OCT1, 0+1/0 Y 4II.1 FLJ43080 PBX1, NKX3A, CDC5, OCT, LUN1, C CDP, NHF1, TST1, CART1, JCA3 chr2 1.95E+08 1.95E+08 12000 ‐0.78699 / SLC39A10 GRE NO Y 5II.1 A JCA5 chr1 57054000 57090000 36000 ‐0.52818 / PCDH17 RP58, OCT1, LHX3, POU6F1, 0+6/0 Y II1A 3 MEISIBHOXA9, HNF1, PBX1, E2F, RORA1, RORA2, NKX61, POU6F1 JCI3 chrX 51845999 51869999 24000 0.33935 SNORA11E / NO 1+0/0 N 5II2 MAGED4B A JCA1 chr8 72786000 72894000 108000 ‐0.78235 EYA1 / MSC BRN2, CHX10, FOX01, MEF2,RP58, 4 / 4 / 0 Y 1III1 SRF, RFX1, CEBP, NFAT, EVI1, A GCNF, CDP, CART1, PPARG,

NKX25, FAC1, HMX1, SEF1, ROAZ 3 2

Table 5. continued JCI3 chr1 57053999 57089999 36000 ‐0.74598 / PCDH17 RP58, OCT1, LHX3, POU6F1, 0+6/0 Y 9II.2 3 MEISIBHOXA9, HNF1, PBX1, E2F, A RORA1, RORA2, NKX61, POU6F1 JCI3 chr5 1.09E+08 1.09E+08 84000 ‐0.56055 MAN2A1 / EVI1, FOXD3, ISRE, FOXJ2, OCT1, 0+1/0 Y 9II.2 FLJ43080 PBX1, NKX3A, CDC5, OCT, LUN1, A CDP, NHF1, TST1, CART1, JCI3 chr6 27737999 27761999 24000 0.57198 ZNF184 / NO 6+5/1 Y 9II.2 HIST1H A

3 3

Table 6. Novel CNVs with regulatory elements containing conserved transcription factor binding sites of factors implicated in renal diseases.

Patient Chr Start Point Stop Point Size of Ratio Adjacent Transcription factor binding ESTs with Conser CNVs Genes site intron/ESTs ved without intron / Region mRNA JCA34II.1C chr1 1.06E+08 1.07E+08 240000 0.54003 / PRMT6 LHX3, HOXA9 6 /14/ 0 Y NTNG1 JCA11III1A chr13 57054000 57090000 36000 ‐0.62275 / PCDH17 OCT1, LHX3, HOXA9, PBX1 0 / 6 / 0 Y JCA15II2C chr13 57042000 57090000 48000 ‐0.64287 / PCDH17 OCT1, LHX3, HOXA9, PBX1 0 / 6 / 0 Y JCA18III.7K chr13 57053999 57089999 36000 ‐0.57056 / PCDH17 OCT1, LHX3, HOXA9, PBX1 0 / 6 / 0 Y JCA22II1A chr13 57054000 57090000 36000 ‐0.62577 / PCDH17 OCT1, LHX3, HOXA9, PBX1 0 / 6 / 0 Y JCA23II4A chr13 57054000 57090000 36000 ‐0.6579 / PCDH17 OCT1, LHX3, HOXA9, PBX1 0 / 6 / 0 Y JCA27II.1A chr13 57053999 57089999 36000 ‐0.59645 / PCDH17 OCT1, LHX3, HOXA9, PBX1 0 / 6 / 0 Y JCA32II.1D chr13 57053999 57101999 48000 ‐0.66508 / PCDH17 OCT1, LHX3, HOXA9, PBX1 0 / 6 / 0 Y JCA5II1A chr13 57054000 57090000 36000 ‐0.52818 / PCDH17 OCT1, LHX3, HOXA9, PBX1 0 / 6 / 0 Y JCI39II.2A chr13 57053999 57089999 36000 ‐0.74598 / PCDH17 OCT1, LHX3, HOXA9, PBX1 0 / 6 / 0 Y JCA14II.1D chr16 88721999 88812636 90637 0.48847 PRDM7 PBX1, HOXA9, PAX2 >100 / >100 Y GAS8/ JCA14II.1F chr5 1.09E+08 1.09E+08 84000 0.30888 MAN2A1 / OCT1, PBX1 0 / 1 / 0 Y FLJ43080 JCA32II.1D chr5 1.09E+08 1.09E+08 84000 ‐0.51128 MAN2A1 / OCT1, PBX1 0 / 1 / 0 Y FLJ43080 JCA34II.1C chr5 1.09E+08 1.09E+08 84000 0.48523 MAN2A1 / OCT1, PBX1 0 / 1 / 0 Y FLJ43080 JCI39II.2A chr5 1.09E+08 1.09E+08 84000 ‐0.56055 MAN2A1 / OCT1, PBX1 0 / 1 / 0 Y FLJ43080 JCA11III1A chr8 72786000 72894000 108000 ‐0.78235 EYA1 / NFAT, FAC1 4 / 4 / 0 y MSC

3 4

Table 7. Transcription Factor genes involved in kidney development.

Genes Type Functions Reference PAX2 Animal Pax2 in mice is responsible for urogenital system Torres et derived development. Pax‐2, as a transcription regulator of the al., 1995 Study paired‐box family, is widely expressed during the (Mouse) development of both ductal and mesenchymal components of the urogenital system. Pax2 homozygous mutant mice lacks kidneys, ureters and genital tracts at birth. This reflects that Pax‐2 is required for multiple steps during the differentiation of intermediate mesoderm. EYA1 Animal An insertion of an intracisternal A particle (IAP) element Johnson et derived into intron 7 of Eya1 gene into mice genome abolished Eya1 al., 1999 Study gene. Expression of Eya1 was reduced consequently due to (Mouse) & formation of aberrant transcripts. The mutant mice develop human a phenotype similar to human branchio‐oto‐renal(BOR) renal syndrome which has also been identified as a results for syndrome EYA1 mutation in human. PBX1 Animal Pbx1 encodes a TALE homeodomain transcription factor Schnabel derived which regulates developmental gene expression. Pbx1 et al., Study homozygous mouse displays abnormal kidneys which has a 2003 (Mouse) reduced size, axially mis‐positioned, and in more severe cases, exhibited unilateral agenesis at E15.5. However, despite the limited expression of Pbx1 in mesenchyme, developing nephrons, and stroma, decreased branching and elongation of the ureter were observed. This reflects that Pbx1 is an essential regulator of mesenchymal function during renal morphogenesis. OCT1 Animal OCT1 encodes an organic ion transporter. During mice Pavlova A derived embryonic development, expression of OCT1 exists in et al., Study midgestation, coinciding with proximal tubule 2000 (Mouse) differentiation in kidneys. it has been observed that expression of OCT1 increased gradually during nephron maturation. LHX1 Animal Lhx1 encodes a LIM‐class homeodomain transcription Akio derived factor that is essential for kidney development. Lhx1 Kobayashi Study expressed in Wolffian Duct, mesonephros and et al., (Mouse) metanephros. Lhx1 lacZ knock‐in allele in mice displays 2004 abnormal pattern by generating an unreported urogenital tissue. Lhx1‐null female mice lack uterus and oviducts while have their ovaries at birth.

36 Table 7. continued HOXA Animal 38 Hox genes encode transcription factors and have crucial Davis AP 11 derived role in determining mammalian body plan. They convey et al., Study regional information along the embryonic axes. The five 1995 (Mouse) most 5' groups (Hox 9‐13) pattern the posterior region of the vertebrate embryo. Mice with both hoxa‐11 and hoxd‐ 11 mutations display a severe kidney defects.

While surveying the novel CNVs we identified in our cases, we were struck by the frequency of one novel CNV (not in the Database of Genomic Variants) on chromosome 13, a recurrent deletion shared by 9 patients. The deletions start from roughly 57,054,000 and is about 36,000 bp long while 2 of them are roughly 48,000bp. The average log 2 ratio is -0.628 which means the ratio of patient DNA to reference DNA is 0.647. None of these patients shares the same pedigree, and 8 of them are from different places around the U.S. with 1 coming from the U.K. 8 of them are Caucasian and 1 is Hispanic / Latino (Table 8.). This indicates that these cases have an infinitesimally small chance of being related to each other and provides strong evidence that this CNV is causative for RA. Because of the deletion’s high frequency, we checked both ends of the deletion to see if any similar repeated sequences might exist as their existence might induce a higher frequency for non-allelic homologous recombination (NAHR). However, by using the UCSC human genome browser, we did not find any particular repeated sequences existing on both sides that stood out from the rest (Figure 5e). Moreover, this deleted region has regions that are highly conserved through vertebrate evolution (Figure 5d) as determined by a 28 species vertebrate comparison which is multiple alignments and two measurements of either conservation across 17 mammal species or conservation of all 28 species. This means it could have potential regulatory significance and have conserved functions in the human genome. Also, there are 6 human ESTs within the region, whichcould be undiscovered genes or novel exons of nearby known genes (Figure 5a). However, these ESTs do not contain introns, making them somewhat less likely to be “real” genes, although further studies are required to rule this out. Although the deletion does not cover any known genes, the regulatory potential score for it

is high because 18 regions reach 0.1(Figure 5c). Regulatory potential(RP) score is computed from alignments of human, chimpanzee , macaque, mouse, rat, dog, and cow. By comparing frequency of short alignment patterns between known regulatory elements and neutral DNA, RP indicates how similar the sequence is compared to alignment pattern typical of regulatory elements. 0.1 of RP represents that there is significant resemblance of sequence to typical regulatory elements. Within these potential regulatory regions, three conserved transcription factor binding sites exist for PBX1, OCT1 and LHX3 (Figure 5b). PBX1 and OCT1 have been shown to be associated with renal disease (Table 7.). LHX3 has been shown to cooperate with LHX1 which is also associated with renal disease) to form heterodimers and both share similar expression patterns during development of motor neurons which later innervate into distinct targets and, probably, kidneys (Jurata LW, 1999). Moreover, Lhx1 and Lhx3 can bind to same binding site which regulate expression of Rxp homeobox gene that functions during gastrulation in the anterior visceral and definitive endoderm. Consequently, due to the deletion of the binding sites in these cases, regulation of target genes controlled by such regulatory elements could be affected and the misregulation of the associated target genes would induce RA. One possible candidate for the target of such regulatory elements is PCDH17, the closest annotated gene. Further experiments of functional analysis would test this hypothesis. Besides this deletion on chromosome 13, 2 of the patients shared a similar deletion on chromosome 5, starting from roughly 109,302,000 and ending at roughly 109,386,000.

Additionally, another 2 cases have amplifications in this region. This region, 84,000 bp, has an average log 2 ratio of -0.536 for deletions and 0.397 for amplifications. At least two of these patients are from different places in the U.S. and have different country origins. One is from New Mexico and a Caucasian, the other is a French Canadian / Caucasian from Arizona. This area on chromosome 5 also has a high regulatory potential score as 18 regions reach 0.1(Figure 6c). Also, several regions are conserved through vertebrate evolution (Figure 6d). The region has one human EST (Figure 6a), and intriguingly, has two conserved binding sites for renal agenesis candidate gene transcription factors: PBX1 and OCT1 (Figure 6b). The OCT1

binding site is particularly interesting in that it sits in the largest region of conservation across the entire genomic interval. As a result, similar to the deletion on chromosome 13, this region, which was found as either an amplification or deletion, is potentially involved in regulation of nearby genes including MAN2A1 and FLJ43080 and thus alteration of such elements might lead to RA. An additional 3 CNVs, though they are not recurrent, still either delete or amplify the binding sites of several transcription factors that are expressed in the kidney, including NFAT, FAC1 and PAX2. Interestingly, PAX2 is directly involved in kidney development. Torres showed that Pax2 homozygous mutant mouse lacks kidneys, ureters and genital tracts due to the dysgenesis of both ductal and mesenchymal components of the developing urogenital system (Torres et al., 1995). Also, patient JCA11III.1A has a deletion on chromosome 8 of 108,000 bp and EYA1 is located just downstream of it. In one mouse study, it was shown that a natural mutation, an insertion of an intracisternal A particle (IAP), disrupted the Eya1 gene in mouse and the mutant mouse developed a syndrome which is similar to branchio-oto-renal (BOR) syndrome in human (Johnson et. Al., 1999). In general, CNVs that do not cover any known genes could still be potentially involved in RA by affecting regulatory elements of relevant genes, or contain new genes or novel exons (of nearby genes) within them. Functional analysis of genes identified through CNV analysis that have been implicated in renal disease (such as EYA1), or specifically expressed in kidneys (such as PBX1), can then be performed in model systems such as frog or zebrafish. Moreover, any region which is highly conserved through vertebrate evolution or contains ESTs or mRNAs is also worthy of functional analysis in these model organisms.

Table 8. Race and origin of members who share similar deletion on chromosome 13.

Patient Chr Start Stop Size of Ratio Race and Origin of Point Point CNVs patient JCA15II.2C chr13 57042000 57090000 48000 ‐0.64287 Caucasian / Connecticut, US JCA11III.1A chr13 57054000 57090000 36000 ‐0.62275 Caucasian / Plymouth, UK JCA5II.1A chr13 57054000 57090000 36000 ‐0.52818 Caucasian / Michigan, US JCA22II.1A chr13 57054000 57090000 36000 ‐0.62577 Caucasian / Massachusetts,US JCA23II.4A chr13 57054000 57090000 36000 ‐0.6579 Caucasian / New Jersey, US JCA27II.1A chr13 57053999 57089999 36000 ‐0.59645 Hispanic, Latino / Michigan, US JCA18III.7K chr13 57053999 57089999 36000 ‐0.57056 Caucasian / Illinois,US JCI39II.2A chr13 57053999 57089999 36000 ‐0.74598 French Canadian, Caucasian / Arizona, US JCA32II.1D chr13 57053999 57101999 48000 ‐0.66508 Caucasian / New Mexico, US

Figure 5. Regulatory elements, ESTs, Conserved region and repeated sequences within chr13: 57,054,000-57,090,000 4 0

Figure 5. (a). Human ESTs within the deletion. (b). Transcription factor binding sites. Red arrows represent transcription factor binding sites that potentially related to RA. (c). 7X regulatory potential (RP) scores computed from alignments of human, chimpanzee (panTro2), macaque (rheMac2), mouse (mm8), rat (rn4), dog (canFam2), and cow (bosTau2). It range from 0 to 0.1. (d). 28 species vertebrate multiz alignment & phastcons conservation. This represent the conservation region of sequences through 28 species vertebrates. (e). Repeated sequences within the region.

Figure 6. Regulatory elements, ESTs, Conserved region and repeated sequences within chr5: 109,301,999-109,385,99 4 2

Figure 6. (a). Human ESTs within the deletion. (b). Transcription factor binding sites. Red arrows represent transcription factor binding sites that potentially related to RA. (c). 7X regulatory potential (RP) scores computed from alignments of human, chimpanzee (panTro2), macaque (rheMac2), mouse (mm8), rat (rn4), dog (canFam2), and cow (bosTau2). It range from 0 to 0.1. (d). 28 species vertebrate multiz alignment & phastcons conservation. This represent the conservation region of sequences through 28 species vertebrates. (e). Repeated sequences within the region.

CONCLUSION AND FUTURE STUDIES

Using aCGH, fifty-seven novel CNVs have been identified by comparing them to the Database of Genomic Variants. Twenty-seven of them contain known genes. BMP4 is one of the genes contained in an amplification in one of the cases and has been shown to be associated with CAKUT while other genes in some of the other CNVs (CNIH, NID2, SPINK5, PTPRK, RHPN2 and CRLF2), though not reported be involved in renal diseases directly, are expressed in kidneys and could thus be associated with RA (Weber s et al., 2008, Tabatabaeifar et al., 2009). For another 30 CNVs that do not cover any known genes, they could be potentially involved in RA by either affecting regulatory elements of RA associated genes or contain new genes or new exons of genes. One recurrent deletion which is shared by 9 patients of different race and from different places contains PBX1, OCT1, and LXH3 transcription factor binding sites. It has already been shown that these genes are associated with nephrogenesis. There are also other CNVs in this category that contain conserved regulatory elements or have renal disease-associated genes upstream or downstream, such as EYA1. Further functional analysis will be done to test some of the genes that have been filtered out in this study. First, known genes that are located within the CNVs and associated with renal disease or specifically expressed in kidneys will be tested by both knockdown and overexpress in Xenopus embryos to determine whether they have any functions in kidney development. Later, genes whose conserved regulatory elements exist in novel CNVs (particularly ones with binding sites for transcription factors expressed in kidney) will be studied by the same methods.

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