BRIEF REVIEW www.jasn.org

Integrating Human and Rodent Data to Identify the Genetic Factors Involved in Chronic Kidney Disease

Michael R. Garrett,* Marcus G. Pezzolesi,† and Ron Korstanje‡ *Department of Medicine and Kidney Disease Center, Medical College of Wisconsin, Milwaukee, Wisconsin; †The Research Division, Joslin Diabetes Center, and Harvard Medical School, Boston, Massachusetts; and ‡The Jackson Laboratory, Bar Harbor, Maine

ABSTRACT The increasing numbers of patients with chronic kidney disease combined with no drome, culminating in renal failure satisfying interventions for preventing or curing the disease emphasize the need to (Supplementary Tables 1 and 2). In these better understand the involved in the initiation and progression of complex cases, there has been strong linkage be- renal diseases, their interactions with other host genes, and the environment. Linkage tween observed phenotypes and specific and association studies in human, rat, and mouse have been successful in identifying chromosomal regions. This approach genetic loci for various disease-related phenotypes but have thus far not been very has yielded several successes, culminat- successful identifying underlying genes. The purpose of this review is to summarize the ing in the identification of mutations in progress in human, rat, and mouse genetic studies to show the concordance between several key genes (NPHS1, NPHS2, the loci among the different species. The collective utilization of human and nonhuman ACTN4, TRPC6, and PLCE1) that play mammalian datasets and resources can lead to a more rapid narrowing of disease loci structural and functional roles in main- and the subsequent identification of candidate genes. In addition, genes identified taining the glomerular filtration bar- through these methods can be further characterized and investigated for interactions rier.14–18 Unraveling the genetics of more using animal models, which is not possible in humans. common forms of kidney disease has been slower to progress, largely because of phe- J Am Soc Nephrol 21: 398–405, 2010. doi: 10.1681/ASN.2009080881 notypic limitations of these studies, their ability to only detect major genetic effects or their reliance on family-based collec- The extent of chronic kidney disease tify those genetic variants that either pre- tions and, perhaps, misconceptions about (CKD) is a growing national concern. Ap- dispose or cause the disease. Evidence of the underlying genetic model contributing proximately 25.6 million people in the familial aggregation and the heritability to their susceptibility; that is, the existence United States exhibit some degree of kid- of biomarkers, such as albuminuria, for of major disease loci and potential - ney injury and/or decline in kidney func- the development and progression of gene and gene-environment interactions. tion.1 Some estimate the number of CKD CKD, as well as various indices of renal More recently, genome-wide associa- patients progressing to end-stage renal dis- function, provide a foundation for inves- tion (GWA) studies, examining as many ease (ESRD) ballooning to more than 2 tigating the genetic predisposition of as 300,000 to 1 million single-nucleotide million in the next 20 years.2 Current treat- CKD.3–12 Efforts to map loci contribut- polymorphisms (SNPs) across the entire ment options are limited and serve only to ing to CKD susceptibility often rely on genome in thousands of unrelated pa- slow progression, not to cure or reverse family-based linkage approaches, which tient and control subjects, are powerful specific conditions. Therefore, under- look to establish statistical associations approaches to detecting disease loci. The standing the genetic basis of kidney disease using genetic markers with a given di- recent success of several GWA studies is of considerable importance because it chotomous (affected versus unaffected) has helped improve our relative under- could provide early diagnosis or more op- or quantitative (GFR or degree of albu- tions for potential targets useful in devel- minuria) phenotypic trait.13 The identi- oping new treatments. fication of genomic locations demon- Published online ahead of print. Publication date strating linkage provides the first line of available at www.jasn.org. evidence suggesting where disease-asso- Correspondence: Dr. Ron Korstanje, The Jackson HUMAN LINKAGE AND GENOME- Laboratory, 600 Main Street, Bar Harbor, ME 04609. ciated genes are likely to reside. The WIDE ASSOCIATION STUDIES Phone: 207-288-6000; Fax: 207-288-6078; E-mail: power of linkage analysis in CKD is best [email protected]

The primary goal of studying the genetics demonstrated by the study of rare famil- Copyright ᮊ 2010 by the American Society of of complex disease in humans is to iden- ial, Mendelian forms of nephrotic syn- Nephrology

398 ISSN : 1046-6673/2103-398 J Am Soc Nephrol 21: 398–405, 2010 www.jasn.org BRIEF REVIEW standing of the genetic basis of complex the largest GWA study of diabetic ne- Project (www.1000genomes.org) prom- human disease. To date, more than 100 phropathy to date, Pezzolesi et al. re- ises to facilitate the identification of such loci for diseases such as coronary heart cently identified associations on chro- variants. Pending its completion, how- disease, type 1 and type 2 diabetes, bipo- mosomes 7p, 9q, 11p, and 13q.24 Lastly, ever, resequencing relevant individuals is lar disorder, Crohn disease, and rheuma- in a GWA study of participants from the still currently required to establish a toid arthritis have been identified using Framingham Heart Study, Hwang et al. comprehensive catalog of potential caus- this approach, providing valuable insight reported associations with albuminuria ative variants for subsequent functional to previously unsuspected biologic path- at loci on 11q and 21q.29 analyses. Finally, current GWA plat- ways and improving our understanding As expected, the odds ratios detected forms poorly assess the contribution of of the allelic architecture that underlies in the majority of GWA studies of CKD rare variants to disease susceptibility, these conditions.19,20 These studies re- have been rather modest, ranging from and analytical methodologies used to veal the effect size of common variants to 1.25 to 1.45—effect sizes far below the evaluate the effects of gene-gene (epi- be more modest than previously sus- threshold detectable using linkage-based static) or gene-environment interactions pected, with odds ratios per risk allele approaches. Despite the early successes on disease remain limited. generally less than 1.4.19 Not surpris- of GWA studies in identifying loci asso- Another result of the HapMap project ingly, GWA studies have proven far su- ciated with CKD, a number of challenges is the identification of markers that differ perior to linkage-based studies, which still lie ahead that must be met to advance between ancestral populations. A novel are powered to identify major loci—loci our understanding of the genetic factors strategy for identifying genes underlying with effect sizes greater than 2.0—in that cause these diseases. At the forefront ancestry-driven diseases, like some identifying genetic variants in common of these challenges is the need for repli- forms of CKD, is mapping by admixture diseases. cation. Although support for several of linkage disequilibrium. Using mapping The promise of GWA approaches has the loci identified by Kottgen et al. and by admixture linkage disequilibrium, recently begun to reap rewards in CKD, Pezzolesi et al. has been demonstrated in both Kopp et al. and Koa et al. identified as five GWA studies have been published cohorts independent of the original asso- strong associations with focal segmental to date (Supplementary Table 3). The ciation signal, additional confirmation is glomerulosclerosis and nondiabetic strongest association identified thus far, necessary to bolster support for these ESRD in African-Americans on chromo- and one of only two reported associa- loci. Currently, a number of efforts are some 22q, and subsequently localized tions to reach a level indicative of “ge- underway to replicate these findings in these associations to the myosin heavy- nome-wide significance,” was reported additional collections, and more GWA chain type II isoform A (MYH9) in a meta-analysis of 41,000 subjects, in- studies in CKD are on the horizon, in- gene.30,31 Although this is a powerful and cluding more than 4300 patients with cluding a study in the Family Investiga- relatively inexpensive method to identify CKD.21 In this study, Kottgen et al. iden- tion of Nephropathy and Diabetes genes that can explain the increased bur- tified significant associations at the uro- (FIND) collection. Continued efforts to den of these diseases among some ethnic modulin (UMOD) locus on chromo- map CKD disease loci, specifically those groups, these genes are most likely only a some 16p for both CKD and the sharing a common genetic mechanism of small subset of the genes involved in the quantitative analysis of GFR. Three modest effect, will rely on meta-analyses disease and can only be applied to very GWA studies have been conducted in pa- of these data. However, such approaches specific cohorts. tients with diabetic nephropathy.22–24 are challenging because of the inherent Variants in the engulfment and cell mo- phenotypic heterogeneity of these collec- tility 1 (ELMO1) gene on tions and the resulting datasets. STUDIES IN RATS AND MICE 7p were initially associated with diabetic Additionally, despite offering far su- IDENTIFY CONCORDANT LOCI nephropathy in a GWA study performed perior resolution in comparison with in a Japanese cohort.22 Variants at this linkage-based approaches (disease loci The rat has been extensively used as a locus have since been reported in two can be localized to regions approxi- model system to study the pathophysiol- large African-American cohorts with mately 5 to 100 kbp in length in GWA ogy and genetics of kidney injury or dis- type 2 diabetes and ESRD and in a collec- studies versus several mega- re- ease,32 including models that also exhibit tion of Caucasian type 1 diabetic patients gions in linkage studies), GWA studies hypertension. A number of well-charac- from the Genetics of Kidneys in Diabetes do not pinpoint the causal functional terized models, including Dahl S, FHH, (GoKinD) study.25,26 Interestingly, this variant. A complete inventory of all cor- MWF, and SBH, have been used for link- same region has also been linked with related variants within the associated re- age analysis of kidney-related traits, in- ESRD and variation in GFR.27,28 A sec- gion, their functional prioritization, and cluding proteinuria, albuminuria, serum ond GWA study in Pima Indians with additional genotyping are required to creatinine, and creatinine clearance. To type 2 diabetes identified a strong associ- fine-map this signal. Together with the date, complete genome scans have been ation in intron 8 of the plasmacytoma HapMap project (www.hapmap.org), performed on 13 experimental crosses, variant translocation (PVT1) gene.23 In the recent launch of the 1000 Genomes identifying more than 40 quantitative

J Am Soc Nephrol 21: 398–405, 2010 Integration of Genetic Data 399 BRIEF REVIEW www.jasn.org trait loci (QTLs) linked to proteinuria 1 2 345 6 alone (Supplemental Table 4). Many of the QTLs have been confirmed and the chromosomal locations further refined using congenic strain analysis,33–40 a method more often used in the rat. Con- genic strains are developed by transfer- ring an entire chromosome (or chromo- somal segment) from one strain onto the genetic background of another through an iterative breeding paradigm that con- tinues until the QTL interval is reduced to contain a relatively small set of genes, 7 8119 10 12 allowing for a detailed analysis of each individual gene contained in the linked region.41 To date, the majority of con- genic rat strains have been developed to study cardiovascular and renal traits and, importantly, such tools have proven vital in further refining the causative disease 42 locus in these models. 13 14 15 16 17 18 Although not as extensively used as the rat, several groups have reported linkage studies in mice over the past few years, mostly using standard inbred strains (C57BL/6J, BALB/c, KK/TaJcl, 129S1/SvImJ, 129S6/SvEvTac, A/J, and 19 20 21 22 X DBA/2J), but also using a hyperlipid- emia-prone Apoe knockout strain43 and the FGS/Kist strain,44 which was selec- tively bred for proteinuria over several generations. Including two reports using strains that are considered models for lu- Human loci (significant) Rat homologous QTL Human GWA pus nephritis,45,46 a total of 18 loci have Human loci (suggestive) Mouse Homologous QTL been mapped for renal damage pheno- types (Supplemental Table 5). Contrary Figure 1. Ideograms of human chromosomes with concordant human, mouse, and rat CKD loci are shown. Human CKD loci identified using linkage (blue) and GWA studies to studies in rat, most of the loci identi- (O-) are indicated on the left side of the chromosome. Concordant loci found in rat fied in mouse are single observations, (green) and mouse (purple) are shown on the right side of the chromosome. with the exception of the distal locus on chromosome 2, which was found in six tween human, rat, and mouse loci, even porate data from animal models, includ- independent crosses. The multiple link- when different renal phenotypes are con- ing both mouse and rat, with those from age analyses performed in rat and mouse sidered, suggests common disease mech- human studies provide valuable insight reveal nearly all chromosomes harbor at anisms link these “subphenotypes” of re- to understanding the causative genes least one renal-related QTL. Interest- nal damage. that underpin CKD, and they offer a ingly, several of these loci are concordant complementary approach to identifying between the two species and, even more the causative disease genes in humans. importantly, many of these loci overlap COMBINING HUMAN, RAT, AND Comparative mapping using combined with homologous regions in humans MOUSE DATA TO NARROW data across species is also a useful tool to (Figure 1). Some QTLs are repeatedly de- GENETIC LOCI narrow loci that are concordant between tected in multiple genetic linkage analy- species. Because of chromosomal rear- ses using different rodent strains, likely There are several approaches one can use rangement between the species, the ho- indicating these loci play more promi- to combining data: mologous regions will not completely nent roles in contributing to renal injury overlap, and the regions that do not relative to other chromosomal regions. Comparative Mapping overlap can be excluded. This approach Moreover, the striking concordance be- Interdisciplinary approaches that incor- was recently used to narrow a locus influ-

400 Journal of the American Society of Nephrology J Am Soc Nephrol 21: 398–405, 2010 www.jasn.org BRIEF REVIEW encing renal function in rat. QTL and genes (Figure 3A). The proximal por- Region-Specific Haplotype Mapping congenic strain analyses localized a QTL tion of this locus is homologous to Most of the inbred strains of mouse and for proteinuria to rat chromosome 2. mouse chromosome 4 and falls within rat are closely related, and parts of their This rat QTL is homologous to both hu- the 95% confidence intervals of the genomes are identical between strains. man chromosomes 1 and 4, with a break- QTL found in the (C57BL/6J ϫ DBA/ Because a QTL can occur only when point occurring near the middle of the 2J)F2 cross51 and the (C57BL/6J ϫ there is variation in the QTL-causing rat QTL (Figure 2). Human studies dem- NZM)F1 ϫ NZM cross.45 The distal gene, we can use this characteristic to onstrate linkage to renal disease on chro- portion of this region is homologous to narrow QTL regions by excluding re- mosome 1,47–49 whereas no linkage has a region on mouse chromosome 13 in gions of the genome where shared haplo- been observed on the homologous re- which no QTL are found. Assuming the types exist between the two strains used gion on chromosome 4. Together, these same underlying gene contributes to for a particular cross. data provided the means to narrow the the association and linkage observed in Considering the human chromosome potential list of candidate genes to focus human and mouse, respectively, all 9q locus described above, this approach on in the rat.50 Another example of com- genes in the interval for which the or- allows further narrowing of the region parative mapping centers on the ne- thologous gene is found on mouse already reduced using comparative map- phropathy locus identified on human chromosome 13 can be excluded as ping. The mouse chromosome 4 region chromosome 9q in the GoKinD popu- likely candidates, leaving only Frmd3 containing Frmd3 and Rasef as candidate lation24 containing 10 potential disease and Rasef as potential disease genes. disease genes was found in two crosses: (C57BL/6JxDBA/2J)F2 and (C57BL/ 6JxNZM)F1 ϫ NZM. When comparing HSA4 haplotypes between B6 and D2, we con-

155703596 FGB clude that B6 and D2 share a haplotype in the region where Rasef is located, and be- RNO2 154921194 SRP2 cause there is no genetic variation be- tween the two strains along this haplo- 174767192 Fgb type, this region could not cause the QTL identified in this cross, and thus we can 175479529 Sfrp2 discount this region as one likely to con- tain the disease genes (Figure 3B). The same is true when comparing C57BL/6J with NZM. Together, this suggests that 151722699 MAB21L2 Frmd3 is the most likely candidate gene 178523415 Mab21l2 179114757 Neph1 HSA1 at this locus. 157580640 D1S484 Another example is the previously mentioned locus on mouse chromosome 2

155983315 D1S2635 found in five different mouse crosses and 180657134 Rab25 REFINED RAT UPE QTL REFINED RAT

OVERLAP OF RAT AND HUMAN RENAL DISEASE LOCI AND HUMAN RENAL OF RAT OVERLAP on the homologous rat chromosome 3 in 180666055 D2Rat230 156229687 NEPH1 two crosses (Supplemental Table 4). If we

182297598 Tpm3 assume it is the same gene underlying these 154897940 D1S2624 QTL, we can combine the mouse data for 154297575 RAB25 haplotype analysis. However, for the 154238965 (C57BL/6J ϫ A/J)F2 and the (C57BL/6J ϫ

CREATININE CLEARANCE-HyperGEN STUDY CREATININE ϫ 152708246 D1S2375 129S1) 129S1 crosses, we observe the B6 152548567 D1S305 AND HYPERTENSION NEPHROPATHY ADULT-ONSET MCKD1 allele as the low allele (low albuminuria 152395457 TPM3 and low Col1 deposits, respectively), Figure 2. Comparative map shows overlap of renal susceptibility loci between rat and whereas in the (C57BL/6J ϫ 129S6)F2 human. The physical map of the rat QTL on chromosome 2 is shown on the left. The cross, we observe the B6 allele as the high proteinuria QTL was successfully narrowed to a small genomic segment through con- allele (increased glomerulosclerosis; Thu 50 genic strain analysis. The region in human that is homologous to the rat QTL lies on Le, personal communication). This sug- both human chromosomes 1 and 4. No linkage for any renal-related traits has been gests in the latter cross the QTL is caused by observed on human chromosome 4 (151 to 155 Mb), whereas linkage with human chromosome 1 (152 to 157 Mb) has been observed in several studies.47,48,60 Taken a different polymorphism. Although together, the region of concordance between the rat and human (1q21) allows the rat 129S1 and 129S6 are related, recent high- QTL to be narrowed by approximately 50%, along with the number of likely candidate resolution genotyping using the Mouse Di- genes. Map distances are in base pairs (www.ensembl.org, Ensembl v38; April 2006). versity Array52 shows regions with large Reproduced with permission from APS.50 variation between the two strains. Com-

J Am Soc Nephrol 21: 398–405, 2010 Integration of Genetic Data 401 BRIEF REVIEW www.jasn.org

A Mmu 4 which could explain the differences be- (C57BL/6J x NZM) F1 x NZM tween these two strains. (C57BL/6J x DBA/2J) F2 When performing haplotype analysis with the strains in the five crosses (after ex- cluding the B6 by 129S6 strain combina- Mouse Chr4 Mouse Chr13 tion), we look for a haplotype that is shared

Rat Chr5 Rat Chr17 by B6, PL, and BALB (the strains providing 80 the resistant allele) and a haplotype that is

RASEF FRMD3 c9orf103 KIF27 RMI1 SLC28A3 shared by NZW, A, KK, DBA/2, and 129S1 6 UBQLN1 60 GKAP1 (the strains providing the susceptible al- HNRPK 4 40 lele). In addition, the haplotypes must be different between the two groups. Only 2 -log10 P-value 20 three small regions on chromosome 2 fit all of these criteria. The first region is approx.

0 0 Recombination rate (cM/Mb) 84,500,000 85,000,000 85,500,000 86,000,000 0.7 Mb large and contains 14 genes, the sec- Position (bp) ond region is 1 Mb and contains 25 genes, and the third region is only 50 kb and con- B 73.1 73.2 73.3 73.4 73.5 73.6 73.7 tains two genes (Figure 4). Here, we can

Rasef Frmd3 also use comparative mapping: the first re- B6 vs DBA/2 gion is concordant to a human QTL for B6 vs NZB/NZW diabetic nephropathy on chromosome 53 Figure 3. Comparative mapping identifies FRMD3 as a novel CKD locus in human and 20p, whereas the other two regions are mouse. (A) The 2-Mb interval on flanking associations identified in the homologous to human chromosome 20q, GWA study of the GoKinD collection24 contains 10 annotated genes. The proximal 1-Mb with no known linkage or association with region is homologous with a region on mouse chromosome 4 that is linked to albuminuria renal phenotypes. Among the 14 genes, QTL that have been mapped in two crosses ((C57BL/6J ϫ DBA/2J/)F2 and (C57BL/6J ϫ only one has a nonsynonymous SNP that NZM)F1 ϫ NZM), whereas the distal 1-Mb region is homologous to a region on mouse matches the strain distribution described chromosome 13 for which no linkage with renal phenotypes has been found. On the basis above. That gene is the P102S change in of the overlapping signal, concordance mapping at this locus implicates only RASEF and Rrbp1, encoding the ribosome-binding FRMD3 as potential candidate CKD genes. (B) QTL mapping detects chromosomal . Gene expression studies are regions that contain genetic variance between the strains used in a particular cross. needed to test the 14 genes for expression Regions that are genetically identical between the two strains, conversely, cannot be linked to the trait of interest. The black bars indicate the regions that are genetically differences among the eight strains. At this different between the parental strains used for the two crosses. Because haplotype point, the above methodology can only be information was not available for NZM, we used data from NZB and NZW, which are the employed in the mouse because the neces- progenitors of NZM. Because RASEF is in a region with no genetic variance in both the sary resources in the rat have not been C57BL/6JxDBA/2J and C57BL/6Jx SM crosses, FRMD3 is the most likely candidate gene developed. Recently, a large-scale SNP dis- for this concordant locus in human and mouse. covery project for the rat (STAR Consor- tium) identified almost 3 million new 130 Mb 140 Mb 150 Mb 160 Mb 170 Mb SNPs, of which 20,238 were evaluated across 167 distinct inbred rat strains and 14 genes 60 genes 13 genes two rat recombinant inbred panels.54 20p 20q These new genetics resources will provide Figure 4. Interval-specific haplotyping and human concordance narrow a locus for the tools to perform similar analysis as de- albuminuria. A QTL for albuminuria was found on the distal part of mouse chromosome scribed for the mouse. Because the QTL in 2 in multiple crosses. Interval-specific haplotyping eliminates the regions in which the two the example described above is also found parental strains for each cross were genetically identical and resulted in three small in the rat, it could confirm the mouse anal- intervals with 14, 60, and 13 genes, respectively. The proximal region is homologous to ysis and maybe even narrow the region human chromosome 20p, for which linkage was found in human, whereas the distal further. region is homologous to human chromosome 20q, for which no linkage has been found. On the basis of the concordance at both the mouse and human loci, the candidate gene is most likely among the 14 genes within the proximal region. ANIMAL MODELS TO TEST CANDIDATE GENES parison of the chromosome 2 region uel de Villena, personal communica- does not show differences between the tion), but it leaves open the possibility As more candidate genes are identified two substrains (Fernando Pardo-Man- of a modifier gene at another locus, by GWA studies in humans, genetic

402 Journal of the American Society of Nephrology J Am Soc Nephrol 21: 398–405, 2010 www.jasn.org BRIEF REVIEW studies in animal models, or a combina- for renal injury in the rat, and first experi- vide valuable insight into our under- tion of both, there will be a need to vali- ments are on the way. standing of causative genes that under- date the candidacy of these genes and es- pin CKD. In this review we have shown tablish a model in which we can explore Transgenics that there is concordance in CKD loci the mechanism and possible interven- The insertion of additional copies of a between species and the possibilities of tion. There are several tools available gene either with its own promoter or narrowing loci by combining human both in mouse and rat, and the choice driven by a strong exogenous promoter and animal data and resources. Com- will depend on the nature of the gene and is possible both in mouse and rat. To bining data will speed up gene discov- the likely effect of found polymorphisms. date, there have been no candidate genes ery that allows us to better understand When it is believed that a dysfunctional for kidney injury identified via linkage the disease mechanisms and identify gene is involved in renal damage, a (con- analysis and subsequently followed up potential targets for intervention. To ditional) knockout model would proba- using a transgenic approach. However, achieve this we need careful annotation bly be the best choice. However, when it there are a number of transgenic models, of each of the loci in the different spe- is overexpression of a gene or a change in such as Tg human renin and/or angio- cies and close collaborations between the protein structure, one might want to tensinogen genes that exhibit increased nephrologists and human and mam- establish a transgenic animal in which hypertension and renal injury56 or Tg malian geneticists. the gene is overexpressed or the gene Ren2,57,58 among many others. Recently, with the altered coding sequence is intro- one group developed a transgenic rat duced. with podocyte-specific expression using ACKNOWLEDGMENTS the human podocin promoter (NPHS2). This study examined the effect of podo- M.R.G. is supported by National Institutes of Knockouts cyte depletion (through Tg diphtheria Health (NIH)/National Heart Lung and Until recently, making a knockout animal toxin receptor) and glomerulosclero- Blood Institute grant HL094446 and funds 59 with null alleles was restricted to mice be- sis. from Advancing Healthier Wisconsin; M.G.P. cause of the need for embryonic stem cells, is supported by NIH grants DK77532 and T32 which are not available for rats. The more DK007260-32; and R.K. is supported by NIH classical approach, in which an is in- CONCLUSION grant DK069381. We thank Joanne Currer for terrupted by a selective marker, is the easi- writing assistance and Jesse Hammer for prep- est but often problematic because the gene Human GWA studies are a powerful aration of the figures. is either involved in development or is cru- new approach for the identification of cial to processes in other organs, leading to loci associated with CKD but require embryonic lethality. The solution to this is large cohorts and subsequent replica- making a conditional knockout. Critical tion. Although these studies have been DISCLOSURES coding are flanked by target “loxP” successful in identifying numerous dis- None. sites susceptible to Cre recombinase. The ease loci, this approach is limited in its reporter-tagged allele can be converted to ability to identify causative variants the lacZ reporter-tagged null allele by ex- and disease genes that underlie these REFERENCES posure to Cre recombinase, causing a associations. 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