BASIC RESEARCH www.jasn.org

Identification of the Nephropathy-Susceptibility Locus HIVAN4

Sindhuri Prakash,* Natalia Papeta,* Roel Sterken,* Zongyu Zheng,* Robert L. Thomas,‡ Zhenzhen Wu,‡ John R. Sedor,‡ Vivette D. DЈAgati,† Leslie A. Bruggeman,‡ and Ali G. Gharavi*

Department of *Medicine and †Pathology, Columbia University College of Physicians and Surgeons, New York, New York 10032, ‡Department of Medicine and the Rammelkamp Center for Education and Research, MetroHealth Medical Center, Case Western Reserve University School of Medicine, Cleveland, Ohio, 44109

ABSTRACT HIVAN1, HIVAN2, and HIVAN3 are nephropathy-susceptibility loci previously identified in the HIV-1 transgenic mouse, a model of collapsing glomerulopathy. The HIVAN1 and HIVAN2 loci modulate expression of Nphs2, which encodes podocin and several other podocyte-expressed . To identify additional loci predisposing to nephropathy, we performed a genome-wide scan in 165 backcross mice generated between the nephropathy-sensitive HIV-1-transgenic FVB/NJ (TgFVB) strain and the resistant Balb/cJ (BALB) strain. We identified a major susceptibility locus (HIVAN4) on 6 G3-F3, with BALB alleles conferring a twofold reduction in severity (peak LOD score ϭ 4.0). Similar to HIVAN1 and HIVAN2, HIVAN4 modulated expression of Nphs2, indicating a common pathway underlying these loci. We independently confirmed the HIVAN4 locus in a sister TgFVB colony that experienced a dramatic loss of nephropathy subsequent to a breeding bottleneck. In this low-penetrance line, 3% of the genome was admixed with BALB alleles, suggesting a remote contamination event. The admixture localized to discrete segments on chromosome 2 and at the HIVAN4 locus. HIVAN4 candidate genes include killer lectin-like genes as well as A2m and Ptpro, whose products are enriched in the glomerulus and interact with HIV-1 . In summary, these data identify HIVAN4 as a major quantitative trait locus for nephropathy and a transregulator of Nphs2. Furthermore, similar selective breeding strategies may help identify further susceptibility loci.

J Am Soc Nephrol 22: 1497–1504, 2011. doi: 10.1681/ASN.2011020209

HIV-1 associated nephropathy (HIVAN) is an icans have identified a major susceptibility locus for AIDS-defining complication of HIV-1 infection. HIVAN, focal segmental glomerulosclerosis, and HIVAN is diagnosed based on kidney biopsy find- nondiabetic kidney failure on chromosome 22, within ings of collapsing focal segmental glomerulosclero- the APOL1 gene.3,4 It is hypothesized that the ne- sis with microcystic tubular dilation.1 In HIVAN, phropathy risk alleles propagated in West Africa be- the normally terminally differentiated glomerular podocytes display dedifferentiation, proliferation, and apoptosis; without antiretroviral therapy, this Received February 28, 2011. Accepted April 13, 2011. disorder frequently results in end-stage kidney fail- Published online ahead of print. Publication date available at ure.1 Patients with HIVAN are almost always of Af- www.jasn.org. rican ancestry and frequently have a strong family Correspondence: Ali G. Gharavi, Department of Medicine, Co- lumbia University College of Physicians and Surgeons, 1150 history of kidney failure, indicating an important Street Nicholas Avenue, Russ Berrie Pavilion Room 412, New role for genetic susceptibility in the pathogenesis of York, New York 10032. Phone: 212-851-5556; Fax: 212-305-5520; disease.2 Consistent with this epidemiology, recent Email: ag2239@ columbia.edu genome-wide association studies in African Amer- Copyright © 2011 by the American Society of Nephrology

J Am Soc Nephrol 22: 1497–1504, 2011 ISSN : 1046-6673/2208-1497 1497 BASIC RESEARCH www.jasn.org

cause the heterozygous genotype confers resistance to trypanoso- ceptibility loci, we generated an HIV1-transgenic backcross by miasis;4 it is not yet clear how homozygosity for these same breeding TgFVB with FVBxBALB F1 mice (52% male). Nearly APOL1 alleles produces susceptibility to histologically diverse ne- one-quarter of the 165 transgenic backcross mice were free of phropathies.5 nephropathy (total renal injury scores Յ3), while the remain- Heterozygous HIV-1 transgenic mice on the FVB/NJ ge- der had varying severity of kidney disease, indicating segrega- netic background (TgFVB) display virtually all of the clinical tion of nephropathy susceptibility alleles in this cross (Supple- and molecular features of HIVAN, enabling dissection of mo- mentary Figure 1a). Consistent with prior descriptions of the lecular pathways leading to disease.6–9 Studies in the murine TgFVB model and previous mapping studies, there were no model have indicated that HIVAN is caused directly by HIV-1 differences in severity of kidney disease based on gender.6,17,18 products, resulting in immune activation in the kidney and a A genome-wide scan and analysis of linkage with 548 infor- hyperplastic response in epithelial cells.6–9 These studies have mative markers identified a major locus on chromosome 6 identified a large number of dysregulated pathways F3-G3, with a peak lod score of 4.0 for the total renal injury (e.g., NF-kB,10,11 MAPK1,2,12,13 Stat312,13) and pathogenic score (peak at rs6329892, 143 Mb, Supplementary Figure 1b). mediators (bFGF,14 VEGF,15 TNF16) that link epithelial prolif- This locus, which we named HIVAN4, achieved genome-wide eration to glomerulosclerosis and are similarly activated in hu- significance based on accepted threshold (Ն3.3) and by 10,000 man disease (reviewed recently in references 7 through 9). The permutations of phenotype on genotype (genome-wide em- nature of the pathogenic process that encompasses these dys- piric p-value ϭ 0.004, Figure 1a). All of the subcomponents of regulated pathways, ultimately leading to proliferation and the histology score yielded peak lod scores of similar magni- scarring of the kidney, is not yet known. However, in conjunc- tude (lod range ϭ 3.7 to 4.9, Figure 1a). This interval was also tion with observations in humans, where very few HIV- significant for BUN (lod ϭ 3.0, empiric P ϭ 0.05) and sugges- infected individuals develop nephropathy, understanding the tive for serum albumin level (lod ϭ 2.4). The lod scores for unique host response to the virus likely holds the key. In this BUN and albumin diminished significantly (lod Ͻ0.5) after regard and similar to humans, genetic susceptibility has a pro- accounting for the histologic injury, indicating that these found effect on the development of nephropathy in the HIV-1 linkages do not represent a direct effect of HIVAN4 on BUN transgenic mouse model: the FVB/NJ strain is very susceptible, and albumin but are mediated by renal injury. Homozygos- but F1 hybrids with BALB/cJ, C57BL/6J and CAST/EiJ are pro- ity for the FVB allele at HIVAN4 conferred a twofold in- tected.17,18 Taking advantage of this strain dependence, we pre- crease in the histologic injury score compared with the het- viously generated mapping cohorts between TgFVB and two erozygote FVB/BALB genotype (Figure 1b). Finally, two counterstrains (C57BL/6J and CAST/EiJ strains), leading to intervals on chromosome 14 (peak lod 1.8 between identification of nephropathy quantitative trait loci (QTLs) on rs13459144 and rs6256423) and chromosome 15 (peak 3, 13, and 4 (HIVAN1, HIVAN2, and HIVAN3 lod ϭ 1.9 at rs13482585) demonstrated suggestive linkage loci, respectively).17,18 Further examination of these loci with for the histologic injury score (Supplementary Figure 1b). expression QTL analysis (expression profiling combined with linkage analysis) showed that the HIVAN1 and HIVAN2 loci HIVAN4 Transregulates Nphs2 Transcript Abundance regulate expression of Nphs2 (encoding Podocin) and several Previous studies have shown that HIVAN1 and HIVAN2 loci other podocyte-expressed genes.18 These data indicated the regulate transcript levels of Nphs2. To determine if HIVAN4 HIVAN1 and HIVAN2 susceptibility genes belong to a com- also transregulates Nphs2 expression, we measured Nphs2 mon regulatory pathway, and they likely introduce genetic le- transcripts abundance in 137 transgenic backcross mice with sions in an expression network involving multiple human ne- available kidney tissue. Nphs2 expression was inversely corre- phropathy genes. lated with renal histology score (r ϭϪ0.62, P ϭ 0.0001), and To identify additional loci predisposing to HIVAN and deter- genome-wide analysis of Nphs2 transcripts abundance re- mine whether Nphs2 transregulation is a common molecular fea- vealed suggestive linkage to the HIVAN4 locus (LOD score 2.1, ture of HIVAN loci, we conducted a new mapping study with the pointwise empiric p-value for linkage to HIVAN4 ϭ 0.006). third resistant counterstrain, Balb/cJ (BALB), leading to the iden- This result was obtained after adjustment for the severity of tification of a new HIVAN QTL. Furthermore, we obtained con- renal injury, indicating that this linkage is not a secondary firmation and higher resolution of this locus in an independent effect of kidney injury but truly represents transregulation. TgFVB colony selectively admixed with BALB alleles. In contrast, there was no evidence of an eQTL for Ptpro ( tryrosine phosphatase, receptor type, O, also known as GLEPP1), which is located within the HIVAN4 locus and en- RESULTS codes a podocyte expressed protein. While Ptpro transcript lev- els was highly correlated with histology injury score (r ϭ Ϫ Mapping a New HIVAN Locus to Chromosome 6 F3-G3 Ϫ0.46, p-value ϭ 3.6 ϫ 10 05) and Nphs2 transcript levels (r ϭ We had previously shown that TgFVBxBALB F1 hybrids do 0.67, P ϭ 2 ϫ 10Ϫ10), there were no differences in transcript not develop nephropathy, suggesting that the BALB genome levels between mice with FVB/FVB and FVB/BALB genotypes, confers resistance to this trait.8 To identify nephropathy sus- after correction for glomerulosclerosis (Lod Ͻ0.5). These find-

1498 Journal of the American Society of Nephrology J Am Soc Nephrol 22: 1497–1504, 2011 www.jasn.org BASIC RESEARCH

Figure 1. (A) Lod plots for the nephropathy traits in the backcross. Map distance is presented on X-axis in Mb. The positions of informative markers are shown above the plot. The dashed horizontal lines indicate the thresholds for significant and suggestive linkage for a backcross based on 10,000 permutations (2.9 and 1.9, respectively). The bar above the lod plot indicates the 95th percentile confidence interval for the HIVAN4 locus. (B) Effect size for glomerulosclerosis and tubular injury score at the peak marker at the HIVAN4 locus. Homozygosity for FVB alleles is associated with a two-fold increase in injury scores.

ings suggest that HIVAN4 alleles do not directly influence homozygous and concordant with FVB alleles at 1292/1305 Ptpro transcript level and that the inverse correlation of Ptpro (99%) of markers across the genome. The 13 SNPs harboring transcript level with glomerulosclerosis occurs secondary to non-FVB alleles were clustered within two distinct intervals on kidney injury. chromosome 2 (85 to 152 Mb between rs3689658 and There were no other loci influencing Nphs2 or Ptpro tran- rs6247960) and chromosome 6 (121 to 139 Mb, between script abundance across the genome; particularly, there was no rs13479006 and rs13479071), and both of these intervals were evidence of an Nphs2 cis-eQTL in this cross, which is consistent fixed to homozygosity in all mice tested (Figure 2C and Sup- with FVB and BALB sharing the same haplotype at the Nphs2 plemental Figure 2). Consistent with the expectation of admix- locus. ture with an albino strain, comparison with reference strains revealed that all 13 non-FVB alleles were derived from the Independent Localization of HIVAN4 in an Admixed BALB genome. Most strikingly, the admixed interval on chro- Colony of TgFVB mosome 6 coincided perfectly with the HIVAN4 locus (Figure Concurrently, in a sister colony of TgFVB located at the Metro- 2C), strongly suggesting that introgression of BALB alleles in Health Medical Center (MHMC), investigators noted a dra- this region accounted for the loss of the nephropathy pheno- matic reduction in the penetrance of renal disease after the type. The lifespan of the low-penetrance TgFVB with the pro- colony was transferred to a new mouse facility in 2006; how- tective BALB alleles was Ͼ9 mo, whereas inbred TgFVB mice ever, two generation of backcrossing to FVB/NJ completely have considerably shorter life spans and require breeding at 6 restored the original nephropathy phenotype excluding trans- to 8 wk of age to sustain the colony (67% die or are moribund gene silencing as a mechanism (Figure 2A). Since the colony at a mean age of 71 d). Since there were no noticeable maintained the albino coat color of the FVB/N background changes in the severity of nephropathy before transfer to the strain, this suggested the occurrence of a spontaneous muta- new facility, this suggested that the contamination existed at tion(s) that conferred resistance to HIVAN or, alternatively, a low level, but BALB alleles rapidly propagated in the new inadvertent introgression of resistance alleles from one or colony because a survival constraint was placed on selection more albino strains. To study this further, the low penetrance of breeders, increasing the use of TgFVB with the protective mice were maintained by brother-sister mating as a separate BALB alleles. Consistent with this prediction, review of colony and 26 mice were genotyped across 1305 SNPs (Illu- breeding records showed that delays during transfer to a mina medium density panel). The low-penetrance mice were new facility in 2006 left older mice with minimal nephrop-

J Am Soc Nephrol 22: 1497–1504, 2011 1499 BASIC RESEARCH www.jasn.org

Figure 2. (A) Breeding scheme in the TgFVB colony at the MetroHealth Medical Center (TgFVB MHMC). Mice ceased to manifest nephropathy after transfer of animals to a new facility in 2006. Histologic examination of mice age Ͼ7 months of age showed normal kidneys (center panel). Brother sister mating (shown on left side) led to the development of a colony showing no kidney disease and significant longevity. However, two generations of backcrossing to FVB/NJ (shown on right side) led to reemergence of early-onset nephropathy (Ͻ2 months of age), as demonstrated by prominent glomerulosclerosis, glomerular collapse, significant tubular dilation and microcysts, cast formation, and interstitial infiltrates. (B) Glomerulosclerosis and tubular injury scores (mean values Ϯ standard errors) in the TgFVB MHMC colony upon initiation of the colony in 2001–2006; a low-penetrance colony was identified after breeding delays during transfer of animals to a new facility in 2006 but the nephropathy phenotype reemerged after backcrossing to FVB/NJ for two generations in 2007. The injury scores in TgFVBxBALB F1 hybrids are shown for comparison. * PϽ0.05. **PϽ0.0001 versus Period 1 or Period 3 (C) Genotypes of the low penetrance colony demonstrate homozygosity for BALB alleles on chromosomes 2 and 6. Each line represents an individual mouse; each square represents genotypes for markers that are informative between FVB (top line, red) and BALB (second line, blue) on the Illumina medium density panel. The area of admixture on chromosome 6 coincides perfectly with the lod-2 interval for the HIVAN4 locus. athy as founders of the new colony, effectively creating a MHMC-TgFVB colony was approximately 35% smaller, genetic bottleneck. spanning a 19-Mb region containing 364 genes (Supple- mentary Table 1). Because a previous mapping study be- Annotation of Positional Candidates Within the tween TgFVB and C57BL/6J (B6) strains did not identify HIVAN4 Locus linkage to HIVAN4,18 this indicated that the FVB and B6 The refined HIVAN4 95th percentile confidence interval strains share the same susceptibility allele at this locus. Con- spans approximately 29 Mb between rs3695724 and sequently, we performed a three-way comparison of haplo- rs3711088 and contains 473 positional candidates. Impor- types, searching for regions within the HIVAN4 interval that tantly, the interval delineated by the low-penetrance are polymorphic between the FVB and BALB strains but are

1500 Journal of the American Society of Nephrology J Am Soc Nephrol 22: 1497–1504, 2011 www.jasn.org BASIC RESEARCH

monomorphic between FVB and B6 strains. In total, 176/ interval-specific congenic and subcongenic lines for identi- 364 genes within the reduced HIVAN4 interval fulfilled fication of causal variants.20 These conditions were inadver- these selection criteria. We further annotated these genes tently recapitulated in the low-penetrance MHMC-TgFVB based on the presence of potentially deleterious amino-acid colony. The mapping resolution was comparable to studies substitutions (16 genes), evidence for interaction of human of outbred populations (approximately 19 Mb at HIVAN4) orthologs with HIV-1 (7 genes), and transcript enrichment and, simultaneously, this colony offered an incipient con- in the glomerulus (8 genes, Supplementary Table 1). Thus, genic strain for rapid generation of HIVAN4 congenic lines. in addition to Ptpro, which was studied further by expres- Our data indicate that selective breeding approaches may sion analysis (as described above), this annotation priori- prove very powerful for localizing and refining murine glo- tizes multiple candidates that can be pursued by sequencing, merulosclerosis susceptibility loci. expression analysis, and functional studies. For example, To date, three crosses with the TgFVB model have iden- the A2m gene, encoding Alpha-2 macroglobulin, harbors a tified four susceptibility loci for HIVAN on chromosomes potentially pathogenic variant, is enriched in the glomeru- 3A1-A3, 13A3-C2, 4A1-A5, and now 6F3-G3 (HIVAN1to4, lus and is predicted to interact with the HIV-1 protease. respectively17,18). We had previously shown that the HI- VAN1 and HIVAN2 loci regulate the expression of multiple podocyte-expressed genes in the absence of HIV-1.18 We DISCUSSION hypothesized that these changes were reac- tive to genetic lesions introduced by the HIVAN risk alleles, In this study, we describe a novel susceptibility locus for suggesting that the murine HIVAN susceptibility genes and nephropathy on mouse chromosome 6 F3-G3 using the podocyte genes belong to the same regulated network. Here HIV-1 transgenic mouse model. This finding was supported we show that HIVAN4 also transregulates Nphs2 expression, by a genome-wide significant detection of this interval in a thereby placing HIVAN1, 2, and 4 loci in the same pathway. backcross cohort derived from inbred strains, and examina- Although these intervals are large, the availability of multi- tion of a low-penetrance colony of TgFVB that demon- ple crosses now enables significant refinement of positional strated selective preservation of the BALB genome at the candidates by comparison of haplotypes among inbred HIVAN4 locus. Since the segments of admixture repre- strains.21 Thus, there are several noteworthy genes among sented Ͻ3% of the mouse genome, it is highly unlikely that the refined list of candidates at the HIVAN4 locus: a cluster the overlap with the HIVAN4 locus is coincidental. The Tg- of natural killer cell lectin-like receptor genes, which encode FVB mice usually have a limited lifespan because they de- proteins important for defense against pathogens,22–26 and velop nephropathy within 3 to 6 wk of age.6,19 This strongly several genes (Bid, A2m, Cd69, Grin2b, Pik3c2g, Plcz1, Itpr2) suggests that the protective BALB alleles at the HIVAN4 with potential interaction with HIV-1. Of note, variation in locus were retained because they conferred a survival and Klra8 gene, encoding a receptor important for natural killer reproductive advantage in the MHMC colony, owing to the cell cytoxicity, is associated with differential susceptibility absence of nephropathy. Moreover, this admixture re- to CMV infection in the mouse but is unlikely to be causal mained undetected due to the albino coat color in the BALB for HIVAN because FVB and BALB have a susceptibility and FVB strains. These findings illustrate the strong effect of haplotype to CMV infection23 Another positional candi- selection on beneficial alleles even within artificial colonies, date, Ptpro, encodes a podocyte expressed protein and has providing further evidence that HIVAN4 exerts a large effect dysregualed expression in HIVAN27,28 but did not demon- on the nephropathy phenotype. A second admixed segment strate a cis-eQTL on additional analysis. Finally, A2m, en- on chromosome 2 was also detected in the low penetrance coding Alpha-2 Macroglobulin, enriched in glomeruli,29 is a MHMC-TgFVB colony and thus represents a candidate lo- target for HIV-1 protease30 and harbors a missense variant cus for nephropathy. The BALB alleles at this locus would be that is predicted to affect protein function. While, to our predicted to have a recessive protective effect because this knowledge, none of the murine HIVAN susceptibility loci interval was not detected in the backcross, which can only overlap with other QTLs for nephropathy or proteinuria in identify additive effects. Further cross breeding can separate the mouse,31–34 the HIVAN4 locus coincides with a QTL for this chromosome-2 interval from the HIVAN4 locus and GFR identified in American Indians.35 Analysis of congenic formally determine whether this region also imparts an ef- lines will enable dissection of specific genes and variants fect on nephropathy. within the HIVAN4 interval, identify relationships to other Our findings indicate that murine nephropathy suscep- QTLs, and delineate common dysregulated pathways down- tibility alleles are under significant selection pressure and, stream of susceptibility alleles. given the opportunity, will be rapidly eliminated from the These studies also offer an interesting parallel to recent gene pool. Many intricate breeding strategies, such as selec- findings demonstrating the effect of natural selection on tive breeding or advanced intercross lines, aim to reproduce nephropathy susceptibility genes in humans. Studies have conditions of selection pressure to localize large-effect shown that genetic variation at the APOL1 locus on chro- QTLs.20 Alternatively, investigators sequentially generate mosome 22 is a major risk factor for nephropathy among

J Am Soc Nephrol 22: 1497–1504, 2011 1501 BASIC RESEARCH www.jasn.org

African Americans with focal segmental glomerulosclerosis purification using the RNeasy kit (QIAGEN). cDNA was generated and nondiabetic end-stage kidney disease3,4 The APOL1 ne- with the Omni-Script kit (Qiagen). Gene expression was measured phropathy risk alleles have rapidly propagated because they in duplicates by quantitative PCR (qPCR) using SYBR-Green mix provide resistance to trypanosomiasis in heterozygotes, but and an iQ5 thermal Cycler (Bio-Rad). Expression values were stan- the increased susceptibility to kidney failure in homozy- dardized to an internal control reference sample (a male FVB/NJ) gotes exerts a balancing effect, maintaining the alternative included in each run, and ␤-actin was used as housekeeping con- alleles in the population.3,4 The mechanism of kidney injury trol (Pfaffl algorithm). Primers for Nphs2 and ␤-actin were previ- for the APOL1 risk-variants is not yet understood. APOL1 is ously described,18 and primers for Ptpro were Ptpro F, 5Ј ÐTGT- only present in hominoids and some Old World monkeys. CATGACGGCCATCCTTA-3Ј, and Ptpro-R, 5Ј-ACTGCGAAGA- About 10 to 15% of healthy African Americans are homozy- GTGACTTCGGA-3Ј. gous for the chromosome 22 risk alleles, and, conversely, 10 to 24% of African Americans with FSGS or hypertensive Genotyping and Analysis of Linkage end-stage kidney failure do not carry any APOL1 risk al- The backcross cohort was genotyped using Illumina medium-den- leles.3,4 These data imply additional genetic or environmen- sity SNP panel, which contains 1449 SNPs distributed across the tal requirements for the development of nephropathy. The genome (Center for Inherited Disease Research genotyping ser- recapitulation of virtually all phenotypic and molecular fea- vice, Baltimore, MD). Of these, 1305 were successfully genotyped ture of human disease in the TgFVB model further suggests and 548 SNPs were informative between FVB and BALB. Linkage that alternative genetic lesions, in the absence of APOL1, scans for histology score, BUN, and serum albumin were per- can predispose to HIVAN. Thus, identification of the mu- formed using R/QTL program with the nonparametric method. rine genetic variants may clarify additional risk factors and Lod scores of Ն3.3 were considered significant.36 In addition, we biologic pathways required for the development of ne- conducted 10,000 permutations of phenotypes on genotype to de- phropathy. termine the empirical significance of the linkage findings. Nphs2 transcript level was square-root transformed to achieve a normal distribution and analyzed using a parametric method (Haley- CONCISE METHODS Knott algorithm) with adjustment for the degree of glomeruloscle- rosis. Pointwise p-value for linkage of the Nphs2 transcript level to the HIVAN4 locus was calculated based on permutations analysis Animal Breeding and Phenotyping All inbred strains were purchased from the Jackson Laborator- limited to the HIVAN4 lod-2 interval. ies (Bar Harbor, ME). The HIV-1 transgenic mouse line TgN(pNL43d14)26Lom (TgFVB) was produced on the inbred Gene Annotation in the HIVAN4 Locus FVB/N genetic background and has been extensively character- SNP identification and annotation across the HIVAN4 interval were ized.6,17 The TgFVB colonies at Columbia University and Metro- obtained from the Mouse Phenome Database (http://phenome.jax. Health Medical Center were established from founders received org/). Segments that were not identical by descent were identified by from the Mount Sinai School of Medicine (courtesy of Dr. Paul filtering for SNPs that are polymorphic between FVB/NJ and BALB/ Klotman). The TgFVB x (FVB x BALB) F1 backcross was generated cByJ, but monomorphic between FVB/NJ and C57BL/6J strains. at Columbia University, and 165 heterozygous HIV-1 transgenic Gene annotation was performed using the PANTHER database progeny were phenotyped at 8 wk of age. Animals that were mor- (http://www.pantherdb.org/). Missense SNPs were evaluated for ibund were euthanized before this end point. Primary phenotypes potential to affect protein function using SIFT (http://blocks.fh- included renal histology, serum albumin, and blood urea nitrogen crc.org/sift/SIFT.html). HIV-1 interactions were obtained from (BUN). Renal histology was scored independently by an investiga- the HIV-1, Human Protein Interaction Database (http://www. tor (VDD) blinded to genetic background and other traits. Three ncbi.nlm.nih.gov/RefSeq/HIVInteractions/). Glomerular enriched genes traits related to tubulointerstitial disease (epithelial regeneration/ were annotated based on a curated list compiled by Lindenmeyer 29 degeneration, tubular casts and dilation, and interstitial infil- et al. trates) and one related to glomerular injury (glomerular sclerosis) were scored using a semiquantitative scale: 0 ϭ no disease; 1 ϭ 1to 25% of tissue showing abnormalities; 2 ϭ 26 to 50%; 3 ϭ 51 to ACKNOWLEDGMENTS 75%; and 4 ϭϾ75% of tissue affected. A global histologic score was calculated as an average of four renal injury scores (range 0 to This study was supported by National Institutes of Health grants 16). The protocol was approved by the IACUC committees at the DK087626 (to AGG) and DK061395 (to LB), and by the MetroHealth Columbia University Medical Center and Case Western Reserve Medical Center institutional funds and the histology core facility of University. the Columbia Diabetes and Endocrinology Research Center (NIH P30-DK63608). RLT was supported by training grant DK007470. Measurement of Podocyte Gene Expression LAB is a member of the Case Center for AIDS Research supported by Total RNA was extracted from mouse whole kidneys using TRIzol grant AI36219. Genotyping services were provided by the Center for reagent (Invitrogen), followed by DNaseI treatment and further Inherited Disease Research (CIDR). CIDR is funded through a federal

1502 Journal of the American Society of Nephrology J Am Soc Nephrol 22: 1497–1504, 2011 www.jasn.org BASIC RESEARCH contract from the National Institutes of Health to The Johns Hopkins TNFR2 interposes the proliferative and NF-kappaB-mediated in- University, contract number HHSN268200782096C. flammatory response by podocytes to TNF-alpha. Lab Invest 91: 413–425, 2011 17. Gharavi AG, Ahmad T, Wong RD, Hooshyar R, Vaughn J, Oller S, Frankel RZ, Bruggeman LA, D’Agati VD, Klotman PE, Lifton RP: DISCLOSURES Mapping a locus for susceptibility to HIV-1-associated nephropathy to mouse chromosome 3. Proc Natl Acad Sci U S A 101: 2488– None. 2493, 2004 18. Papeta N, Chan KT, Prakash S, Martino J, Kiryluk K, Ballard D, Bruggeman LA, Frankel R, Zheng Z, Klotman PE, Zhao H, D’Agati VD, Lifton RP, Gharavi AG: Susceptibility loci for murine HIV-associated nephropathy encode trans- REFERENCES regulators of podocyte gene expression. J Clin Invest 119: 1178–1188, 2009 19. Chan KT, Papeta N, Martino J, Zheng Z, Frankel RZ, Klotman PE, 1. Wyatt CM, Klotman PE: HIV-1 and HIV-associated nephropathy 25 D’Agati VD, Lifton RP, Gharavi AG: Accelerated development of years later. Clin J Am Soc Nephrol 2: S20–24, 2007 collapsing glomerulopathy in mice congenic for the HIVAN1 locus. 2. Freedman BI, Soucie JM, Stone SM, Pegram S: Familial clustering Kidney Int 75: 366–372, 2009 of end-stage renal disease in blacks with HIV-associated nephrop- 20. Darvasi A: Experimental strategies for the genetic dissection of athy. Am J Kidney Dis 34: 254–258, 1999 complex traits in animal models. Nat Genet 18: 19–24, 1998 3. Genovese G, Tonna SJ, Knob AU, Appel GB, Katz A, Bernhardy AJ, 21. Wade CM, Kulbokas EJ, 3rd, Kirby AW, Zody MC, Mullikin JC, Lander Needham AW, Lazarus R, Pollak MR: A risk allele for focal segmen- ES, Lindblad-Toh K, Daly MJ: The mosaic structure of variation in the tal glomerulosclerosis in African Americans is located within a genome. Nature 420: 574–578, 2002 region containing APOL1 and MYH9. Kidney Int 78: 698–704, 2010 22. Lee SH, Girard S, Macina D, Busa M, Zafer A, Belouchi A, Gros P, 4. Genovese G, Friedman DJ, Ross MD, Lecordier L, Uzureau P, Vidal SM: Susceptibility to mouse cytomegalovirus is associated Freedman BI, Bowden DW, Langefeld CD, Oleksyk TK, Uscinski with deletion of an activating natural killer cell receptor of the Knob AL, Bernhardy AJ, Hicks PJ, Nelson GW, Vanhollebeke B, C-type lectin superfamily. Nat Genet 28: 42–45, 2001 Winkler CA, Kopp JB, Pays E, Pollak MR: Association of trypanolytic 23. Lee SH, Gitas J, Zafer A, Lepage P, Hudson TJ, Belouchi A, Vidal ApoL1 variants with kidney disease in African Americans. Science SM: Haplotype mapping indicates two independent origins for the 329: 841–845, 2010 Cmv1s susceptibility allele to cytomegalovirus infection and refines 5. Freedman BI, Kopp JB, Langefeld CD, Genovese G, Friedman DJ, its localization within the Ly49 cluster. Immunogenetics 53: 501– Nelson GW, Winkler CA, Bowden DW, Pollak MR: The apolipopro- 505, 2001 tein L1 (APOL1) gene and nondiabetic nephropathy in African 24. Delano ML, Brownstein DG: Innate resistance to lethal mousepox is Americans. J Am Soc Nephrol 21: 1422–1426, 2010 genetically linked to the NK gene complex on chromosome 6 and 6. Kopp JB, Klotman ME, Adler SH, Bruggeman LA, Dickie P, Marinos correlates with early restriction of virus replication by cells with an NJ, Eckhaus M, Bryant JL, Notkins AL, Klotman PE: Progressive NK phenotype. J Virol 69: 5875–5877, 1995 glomerulosclerosis and enhanced renal accumulation of basement 25. Idris AH, Smith HR, Mason LH, Ortaldo JR, Scalzo AA, Yokoyama membrane components in mice transgenic for human immunode- WM: The natural killer gene complex genetic locus Chok encodes ficiency virus type 1 genes. Proc Natl Acad SciUSA89: 1577– Ly-49D, a target recognition receptor that activates natural killing. 1581, 1992 Proc Natl Acad Sci U S A 96: 6330–6335, 1999 7. Rosenstiel P, Gharavi A, D’Agati V, Klotman, P. Transgenic and 26. Parham P, Abi-Rached L, Matevosyan L, Moesta AK, Norman PJ, infectious animal models of HIV-associated nephropathy. JAmSoc Older Aguilar AM, Guethlein LA: Primate-specific regulation of Nephrol 20: 2296–2304, 2009 natural killer cells. J Med Primatol 39: 194–212, 2010 8. Bruggeman LA, Nelson PJ: Controversies in the pathogenesis of 27. Barisoni L, Kriz W, Mundel P, & D’Agati, V.: The dysregulated HIV-associated renal diseases. Nat Rev Nephrol 5: 574–581, 2009 podocyte phenotype: a novel concept in the pathogenesis of col- 9. Albaqumi M, Soos TJ, Barisoni L, Nelson PJ: Collapsing glomeru- lapsing idiopathic focal segmental glomerulosclerosis and HIV- lopathy. J Am Soc Nephrol 17: 2854–2863, 2006 associated nephropathy. J Am Soc Nephrol 10: 51–61, 1999 10. Bruggeman LA, Adler SH, Klotman PE: Nuclear factor-kappa B 28. Wharram BL, Goyal M, Gillespie PJ, Wiggins JE, Kershaw DB, binding to the HIV-1 LTR in kidney: Implications for HIV-associated Holzman LB, Dysko RC, Saunders TL, Samuelson LC, Wiggins RC: nephropathy. Kidney Int 59: 2174–2181, 2001 Altered podocyte structure in GLEPP1 (Ptpro)-deficient mice asso- 11. Ross MJ, Martinka S, D’Agati VD, Bruggeman LA: NF-kappaB reg- ciated with hypertension and low glomerular filtration rate. J Clin ulates Fas-mediated apoptosis in HIV-associated nephropathy. Invest 106: 1281–1290, 2000 J Am Soc Nephrol 16: 2403–2411, 2005 29. Lindenmeyer MT, Eichinger F, Sen K, Anders HJ, Edenhofer I, 12. He JC, Husain M, Sunamoto M, D’Agati VD, Klotman ME, Iyengar Mattinzoli D, Kretzler M, Rastaldi MP, Cohen CD: Systematic anal- R, Klotman PE: Nef stimulates proliferation of glomerular podo- ysis of a novel human renal glomerulus-enriched gene expression cytes through activation of Src-dependent Stat3 and MAPK1,2 dataset. PLoS One 5: e11545, 2010 pathways. J Clin Invest 114: 643–651, 2004 30. Meier UC, Billich A, Mann K, Schramm HJ, Schramm W: alpha 13. Lu TC, Wang ZH, Feng X, Chuang PY, Fang W, Shen Y, Levy DE, 2-Macroglobulin is cleaved by HIV-1 protease in the bait region but Xiong H, Chen N, He JC: Knockdown of Stat3 activity in vivo not in the C-terminal inter-domain region. Biol Chem Hoppe Seyler prevents diabetic glomerulopathy. Kidney Int 76: 63–71, 2009 372: 1051–1056, 1991 14. Ray PE, Bruggeman LA, Weeks BS, Kopp JB, Bryant JL, Owens JW, 31. Chua S, Jr., Li Y, Liu SM, Liu R, Chan KT, Martino J, Zheng Z, Susztak Notkins AL, Klotman PE: bFGF and its low affinity receptors in the K, D’Agati VD, Gharavi AG: A susceptibility gene for kidney disease pathogenesis of HIV-associated nephropathy in transgenic mice. in an obese mouse model of type II diabetes maps to chromosome Kidney Int 46: 759–772, 1994 8. Kidney Int 78: 453–462, 2010 15. Korgaonkar SN, Feng X, Ross MD, Lu TC, D’Agati V, Iyengar R, 32. Hageman RS, Leduc MS, Caputo CR, Tsaih SW, Churchill GA, Klotman PE, He JC: HIV-1 upregulates VEGF in podocytes. JAm Korstanje, R. Uncovering genes and regulatory pathways related to Soc Nephrol 19: 877–883, 2008 urinary albumin excretion. J Am Soc Nephrol 22: 73–81, 2011 16. Bruggeman LA, Drawz PE, Kahoud N, Lin K, Barisoni L, Nelson PJ: 33. Sheehan S, Tsaih SW, King BL, Stanton C, Churchill GA, Paigen B,

J Am Soc Nephrol 22: 1497–1504, 2011 1503 BASIC RESEARCH www.jasn.org

DiPetrillo K: Genetic analysis of albuminuria in a cross between merular filtration rate in American Indians. Kidney Int 74: 1185– C57BL/6J and DBA/2J mice. Am J Physiol Renal Physiol 293: 1191, 2008 F1649–1656, 2007 36. Lander E, Kruglyak L: Genetic dissection of complex traits: guide- 34. Ratelade J, Lavin TA, Muda AO, Morisset L, Mollet G, Boyer O, lines for interpreting and reporting linkage results. Nat Genet 11: Chen DS, Henger A, Kretzler M, Hubner N, Thery C, Gubler MC, 241–247, 1995 Montagutelli X, Antignac C, Esquivel EL: Maternal environment interacts with modifier genes to influence progression of nephrotic syndrome. J Am Soc Nephrol 19: 1491–1499, 2008 35. Mottl AK, Vupputuri S, Cole SA, Almasy L, Goring HH, Diego VP, Laston S, Franceschini N, Shara NM, Lee ET, Best LG, Fabsitz Supplemental information for this article is available online at http://www.jasn. RR, MacCluer JW, Umans JG, North KE: Linkage analysis of glo- org/.

1504 Journal of the American Society of Nephrology J Am Soc Nephrol 22: 1497–1504, 2011 Supplementary figure 1. A. Distribution of the total injury score in the backcross. B. Genome‐wide lod plot of the total injury score

A B

Supplementary Figure 2 Genome­wide distrubution of genotypes in 26 MHMC­TgFVB mice. Each row represents a mouse, each column a marker. Markers are grouped by marker FVB alleles are presented in red, BALB alleles in blue (autosomes) or green (X‐chromosome). The first and second row represent genotypes from an FVB/NJ and an Balb/cJ mouse. Only two intervals show homozygosity for Balb/cJ alleles.

Supplementary table. Genes located within compatible haploype blocks in the HIVAN4 lod-2 interval (the interval delineated by the admixed strain is shown in blue)

Location HIV- Nonsynonymous Damaging Damaging Gene Symbol (Mb) Description Description interaction? Glomerular enriched? variant? variant? SNP ID Wnk1 119.873545 MGI:2442092 WNK lysine deficient protein kinase 1 - - - - - Ninj2 120.041726 MGI:1352751 ninjurin 2 - - - - - B4galnt3 120.15186 MGI:3041155 beta-1,4-N-acetyl-galactosaminyl transferase 3 - - - - - Jarid1a 120.310673 MGI:2136980 jumonji, AT rich interactive domain 1A (Rbp2 like) - - - - - Il17ra 120.411546 MGI:107399 interleukin 17 receptor A - - YES - - Cecr5 120.457527 MGI:2136976 cat eye syndrome chromosome region, candidate 5 homolog (human) - - - - - 1700072O05Rik 120.502874 MGI:1920755 RIKEN cDNA 1700072O05 gene - - - - - Cecr2 120.614746 MGI:1923799 cat eye syndrome chromosome region, candidate 2 homolog (human) - - - - - Slc25a18 120.721731 MGI:1919053 solute carrier family 25 (mitochondrial carrier), member 18 - - - - - Atp6v1e1 120.743344 MGI:894326 VATPase, H+ transporting, lysosomal V1 subunit E1 - - - - - Bcl2l13 120.784809 MGI:2136959 BCL2-like 13 (apoptosis facilitator) - - YES - - Bid 120.841357 MGI:108093 BH3 interacting domain death agonist YES - - - - BC030863 120.881725 MGI:2673704 cDNA sequence BC030863 - - - - - Mical3 120.951381 MGI:2442733 microtubule associated monoxygenase, calponin and LIM domain containing 3 - - YES - - LOC100043793 121.012523 Entrez100043793 - - - YES - - Pex26 121.133685 MGI:1921293 peroxisome biogenesis factor 26 - - - - - Usp18 121.194084 MGI:1344364 ubiquitin specific peptidase 18 - - - - - Slc6a13 121.248356 MGI:95629 solute carrier family 6 (neurotransmitter transporter, GABA), member 13 - - - - - Iqsec3 121.321361 MGI:2677208 IQ motif and Sec7 domain 3 - - - - - A2m 121.576011 MGI:2449119 alpha-2-macroglobulin YES YES YES YES rs50350755 Mug1 121.786989 MGI:99837 murinoglobulin 1 - - - - - Mug2 121.956816 MGI:99836 murinoglobulin 2 - - YES - - LOC677375 122.395336 Entrez677375 ------4933426K21Rik 122.402698 MGI:1918325 RIKEN cDNA 4933426K21 gene - - - - - Mfap5 122.461859 MGI:1354387 microfibrillar associated protein 5 - YES - - - Aicda 122.501832 MGI:1342279 activation-induced cytidine deaminase YES - - - - Apobec1 122.52683 MGI:103298 apolipoprotein B editing complex 1 - - - - - Gdf3 122.553968 MGI:95686 growth differentiation factor 3 - - - - - Dppa3 122.574467 MGI:1920958 developmental pluripotency-associated 3 - - - - - Clec4a4 122.938533 MGI:3624119 C-type lectin domain family 4, member a4 - - - - - Clec4b1 122.998101 MGI:1917060 C-type lectin domain family 4, member b1 - - YES - - EG546913 123.559776 Entrez546913 ------EG545874 123.771406 Entrez545874 - - - YES - - V2r1b 123.973582 Entrez56552 ------Vmn2r26 123.974776 MGI:2678394 vomeronasal 2, receptor 26 - - - - - Cd163 124.254532 MGI:2135946 CD163 antigen - - - - - C1r 124.461202 MGI:1355313 complement component 1, r subcomponent - - - - - Ncapd2 125.116061 MGI:1915548 non-SMC condensin I complex, subunit D2 - - - - - Tmem16b 125.658841 MGI:2387214 transmembrane protein 16B - - - - - Parp11 127.401896 MGI:2141505 poly (ADP-ribose) polymerase family, member 11 - - - - - Efcab4b 127.526039 MGI:2685919 EF-hand calcium binding domain 4B - - - - - Prmt8 127.637041 MGI:3043083 protein arginine N-methyltransferase 8 - - - - - Tspan11 127.835693 MGI:1915748 tetraspanin 11 - - YES - - Tspan9 127.910308 MGI:1924558 tetraspanin 9 - - - - - ENSMUSG00000056771 128.137864 MGI:3641978 predicted gene, ENSMUSG00000056771 - - - - - Tead4 128.175172 MGI:106907 TEA domain family member 4 - - - - - Tulp3 128.269499 MGI:1329045 tubby-like protein 3 - - - - - Itfg2 128.358417 MGI:1915450 integrin alpha FG-GAP repeat containing 2 - - - - - Pzp 128.431673 MGI:87854 pregnancy zone protein - - - - - BC048546 128.488385 MGI:3039594 cDNA sequence BC048546 - - - - - Klrb1a 128.559225 MGI:107540 killer cell lectin-like receptor subfamily B member 1A - - YES - - LOC677440 128.598277 Entrez677440 ------Clec2h 128.610407 MGI:2136934 C-type lectin domain family 2, member h - - YES - - LOC100043863 128.62405 Entrez100043863 - - - YES - - LOC100043861 128.656526 Entrez100043861 - - - YES - - Klrb1c 128.726546 MGI:107538 killer cell lectin-like receptor subfamily B member 1C - - YES - - Klrb1b 128.728507 MGI:107539 killer cell lectin-like receptor subfamily B member 1B - - YES - - LOC667706 128.83385 Entrez667706 ------Clec2i 128.835633 MGI:2136650 C-type lectin domain family 2, member i - - - - - Clec2g 128.882408 MGI:1918059 C-type lectin domain family 2, member g - - YES - - LOC667715 128.926519 Entrez667715 ------BC064078 128.942979 MGI:3040692 cDNA sequence BC064078 - - - - - Clec2f 128.964152 MGI:3522133 C-type lectin domain family 2, member f - - YES - - Klrb1f 128.993971 MGI:2442965 killer cell lectin-like receptor subfamily B member 1F - - - - - 4922502D21Rik 129.270245 MGI:2685920 RIKEN cDNA 4922502D21 gene - - - - - Clec12a 129.299402 MGI:3040968 C-type lectin domain family 12, member a - - - - - Clec1a 129.374717 MGI:2444151 C-type lectin domain family 1, member a - - - - - Clec7a 129.409864 MGI:1861431 C-type lectin domain family 7, member a - - YES - - Olr1 129.433371 MGI:1261434 oxidized low density lipoprotein (lectin-like) receptor 1 - - - - - D630042F21Rik 129.460607 MGI:2442838 RIKEN cDNA D630042F21 gene - - - - - Klrk1 129.558366 MGI:1196250 killer cell lectin-like receptor subfamily K, member 1 - - - - - Klrc3 129.587119 MGI:1929720 killer cell lectin-like receptor subfamily C, member 3 - - - - - Klrc2 129.596062 MGI:1336162 killer cell lectin-like receptor subfamily C, member 2 - - - - - Klrc1 129.616039 MGI:1336161 killer cell lectin-like receptor subfamily C, member 1 - - - - - Klri1 129.645237 MGI:3530275 killer cell lectin-like receptor family I member 1 - - YES - - Klri2 129.677163 MGI:2443965 killer cell lectin-like receptor family I member 2 - - YES - - Gm156 129.714753 MGI:2685002 gene model 156, (NCBI) - - YES YES rs46035388 Klra17 129.779204 MGI:2180674 killer cell lectin-like receptor, subfamily A, member 17 - - YES YES rs36722353, rs36828428, rs37223080, rs37236681 Klra5 129.847056 MGI:101903 killer cell lectin-like receptor, subfamily A, member 5 - - YES YES rs37892132 Klra6 129.961162 MGI:101902 killer cell lectin-like receptor, subfamily A, member 6 - - YES YES rs38914815, rs37814296 LOC100038893 129.961162 Entrez100038893 - - - YES - - Klra19 129.963051 MGI:2136936 killer cell lectin-like receptor, subfamily A, member 19 - - YES YES rs38914815, rs37814296 LOC100038894 129.99177 Entrez100038894 - - - YES - - LOC100038895 129.99177 Entrez100038895 ------Klra4 129.992222 MGI:101904 killer cell lectin-like receptor, subfamily A, member 4 - - YES - - Klra16 129.99375 MGI:1351669 killer cell lectin-like receptor, subfamily A, member 16 - - YES - - EG654449 130.032616 MGI:3644243 predicted gene, EG654449 - - YES - - Klra14 130.065237 MGI:1321089 killer cell lectin-like receptor subfamily A, member 14 - - - - - Klra9 130.126879 MGI:1321153 killer cell lectin-like receptor subfamily A, member 9 - - YES - - Klra7 130.166698 MGI:101901 killer cell lectin-like receptor, subfamily A, member 7 - - - - - LOC100038898 130.21416 Entrez100038898 ------Klra12 130.214482 MGI:1321091 killer cell lectin-like receptor subfamily A, member 12 - - YES - - Klra10 130.217292 MGI:1321093 killer cell lectin-like receptor subfamily A, member 10 - - YES YES rs38861679 Klra13 130.239327 MGI:1321090 killer cell lectin-like receptor subfamily A, member 13 - - YES - - Klra3 130.271914 MGI:101905 killer cell lectin-like receptor, subfamily A, member 3 - - - - - Klra1 130.313936 MGI:101907 killer cell lectin-like receptor, subfamily A, member 1 - - YES - - LOC100038902 130.313937 Entrez100038902 ------LOC100038903 130.313942 Entrez100038903 - - - YES - - EG667798 130.615388 MGI:3648641 predicted gene, EG667798 - - YES - - EG625558 130.678446 MGI:3643376 predicted gene, EG625558 - - - - - EG636947 130.750525 MGI:3648824 predicted gene, EG636947 - - YES - - EG625648 130.936083 MGI:3645795 predicted gene, EG625648 - - - - - EG434094 131.113318 MGI:3643462 predicted gene, EG434094 - - - - - Klra2 131.167499 MGI:101906 killer cell lectin-like receptor, subfamily A, member 2 - - YES - - 2010012C16Rik 131.23243 MGI:1913691 RIKEN cDNA 2010012C16 gene - - - - - Styk1 131.247319 MGI:2141396 serine/threonine/tyrosine kinase 1 - - YES - - Csda 131.312986 MGI:2137670 cold shock domain protein A - YES - - - LOC625758 131.434468 Entrez625758 ------5430401F13Rik 131.492175 MGI:1918642 RIKEN cDNA 5430401F13 gene - - YES - - LOC625781 131.556525 Entrez625781 - - - YES YES rs45775618, rs46068285, rs51503960, rs46783363 Tas2r107 131.608537 MGI:2681207 taste receptor, type 2, member 107 - - YES - - Tas2r106 131.6264 MGI:2681203 taste receptor, type 2, member 106 - - - - - Tas2r104 131.633142 MGI:2681185 taste receptor, type 2, member 104 - - - - - Tas2r105 131.634753 MGI:2681195 taste receptor, type 2, member 105 - - YES YES rs49780685, rs47147082 Tas2r114 131.637229 MGI:2681218 taste receptor, type 2, member 114 - - YES YES rs30481322 EG114600 132.064239 MGI:3647826 predicted gene, EG114600 - - - - - Prb1 132.154845 MGI:2681872 proline-rich protein BstNI subfamily 1 - - - - - LOC631126 132.156064 Entrez631126 ------EG667929 132.309477 MGI:3645847 predicted gene, EG667929 - - YES - - EG381818 132.388549 MGI:3646668 predicted gene, EG381818 - - - - - Prh1 132.517963 MGI:97773 proline rich protein HaeIII subfamily 1 - - - - - Prp2 132.544052 MGI:1932491 proline rich protein 2 - - YES - - A630073D07Rik 132.574746 MGI:2686534 RIKEN cDNA A630073D07 gene - - - - - Tas2r120 132.605873 MGI:2681256 taste receptor, type 2, member 120 - - - - - Tas2r121 132.64817 MGI:2681259 taste receptor, type 2, member 121 - - YES - - Tas2r122 132.659069 MGI:2681262 taste receptor, type 2, member 122 - - YES - - Tas2r115 132.685276 MGI:2681223 taste receptor, type 2, member 115 - - YES YES rs30805791, rs13479040 Tas2r124 132.703112 MGI:2681267 taste receptor, type 2, member 124 - - - - - Tas2r102 132.710199 MGI:2681171 taste receptor, type 2, member 102 - - YES YES rs31839244, rs31838241, rs31845133, rs31845773 Tas2r117 132.750982 MGI:2681242 taste receptor, type 2, member 117 - - - - - Tas2r123 132.795245 MGI:2681264 taste receptor, type 2, member 123 - - YES - - Tas2r116 132.803543 MGI:1890258 taste receptor, type 2, member 116 - - YES - - Tas2r110 132.816162 MGI:2681216 taste receptor, type 2, member 110 - - YES - - Tas2r113 132.841232 MGI:2681217 taste receptor, type 2, member 113 - - YES - - Location HIV- Nonsynonymous Damaging Damaging Gene Symbol (Mb) Description Description interaction? Glomerular enriched? variant? variant? SNP ID Tas2r125 132.857902 MGI:2681269 taste receptor, type 2, member 125 - - YES YES rs30119082 Tas2r129 132.899141 MGI:2681276 taste receptor, type 2, member 129 - - YES YES rs29873427 Tas2r131 132.905062 MGI:2681280 taste receptor, type 2, member 131 - - YES YES rs31849649, rs31851504 Tas2r109 132.928045 MGI:2681214 taste receptor, type 2, member 109 - - YES - - Tas2r140 133.002941 MGI:2681298 taste receptor, type 2, member 140 - - YES - - 2700089E24Rik 133.052061 MGI:1914379 RIKEN cDNA 2700089E24 gene - - - - - LOC630618 133.063306 Entrez630618 ------5530400C23Rik 133.240376 MGI:1918650 RIKEN cDNA 5530400C23 gene - - - - - EG545884 133.477274 MGI:3648672 predicted gene, EG545884 - - YES - - LOC100043653 133.680646 Entrez100043653 - - - YES YES rs31885095, rs30857367 EG545886 133.706227 MGI:3648674 predicted gene, EG545886 - - YES - - LOC622962 133.710839 Entrez622962 ------Kap 133.797873 MGI:96653 kidney androgen regulated protein - - YES - - LOC668063 133.881605 Entrez668063 ------Etv6 133.983802 MGI:109336 ets variant gene 6 (TEL oncogene) - - - - - Bcl2l14 134.344356 MGI:1914063 Bcl2-like 14 (apoptosis facilitator) - - - - - Lrp6 134.398236 MGI:1298218 low density lipoprotein receptor-related protein 6 - - YES - - Mansc1 134.557389 MGI:1914979 MANSC domain containing 1 - YES YES - - Loh12cr1 134.589009 MGI:1915024 loss of heterozygosity, 12, chromosomal region 1 homolog (human) - - - - - Dusp16 134.663557 MGI:1917936 dual specificity phosphatase 16 - - - - - Crebl2 134.778286 MGI:1889385 cAMP responsive element binding protein-like 2 - - - - - Gpr19 134.817289 MGI:892973 G protein-coupled receptor 19 - - - - - 1190002F15Rik 134.877474 MGI:1915081 RIKEN cDNA 1190002F15 gene - - - - - Apold1 134.930107 MGI:2685921 apolipoprotein L domain containing 1 - - YES - - Ddx47 134.959815 MGI:1915005 DEAD (Asp-Glu-Ala-Asp) box polypeptide 47 - - - - - Gprc5d 135.054029 MGI:1935037 G protein-coupled receptor, family C, group 5, member D - - - - - Hebp1 135.085537 MGI:1333880 heme binding protein 1 - YES - - - 8430419L09Rik 135.146107 MGI:1921775 RIKEN cDNA 8430419L09 gene - - - - - Gsg1 135.185412 MGI:1194499 germ cell-specific gene 1 - - YES - - LOC100043890 135.2972 Entrez100043890 ------Emp1 135.311049 MGI:107941 epithelial membrane protein 1 - - - - - Grin2b 135.677995 MGI:95821 glutamate receptor, ionotropic, NMDA2B (epsilon 2) YES - - - - E330021D16Rik 136.350364 MGI:2141773 RIKEN cDNA E330021D16 gene - - - - - Atf7ip 136.465549 MGI:1858965 activating 7 interacting protein - - - - - 1100001H23Rik 136.558744 MGI:1914107 RIKEN cDNA 1100001H23 gene - - - - - Gucy2c 136.643825 MGI:106903 2c - - - - - Hist4h4 136.751204 MGI:2448443 histone cluster 4, H4 YES - - - - H2afj 136.754941 MGI:3606192 H2A histone family, member J - - - - - Wbp11 136.760202 MGI:1891823 WW domain binding protein 11 - - - - - BC049715 136.775493 MGI:3605234 cDNA sequence BC049715 - - - - - C030030A07Rik 136.776816 MGI:2443451 RIKEN cDNA C030030A07 gene - - YES - - Art4 136.795377 MGI:1202710 ADP-ribosyltransferase 4 - - - - - Mgp 136.819382 MGI:96976 matrix Gla protein - YES - - - Erp27 136.853913 MGI:1916437 endoplasmic reticulum protein 27 - - - - - Arhgdib 136.870268 MGI:101940 Rho, GDP dissociation inhibitor (GDI) beta - YES - - - Pde6h 136.901132 MGI:1925850 phosphodiesterase 6H, cGMP-specific, cone, gamma - - - - - Rerg 137.001383 MGI:2665139 RAS-like, estrogen-regulated, growth-inhibitor - - - - - Ptpro 137.199012 MGI:1097152 protein tyrosine phosphatase, receptor type, O YES YES - - - Ptpn15 137.200986 MGI:104711 protein tyrosine phosphatase, non-receptor type 15 - - - - - Lmo3 138.312745 MGI:102810 LIM domain only 3 - - - - - Igbp1b 138.603666 MGI:1354380 immunoglobulin (CD79A) binding protein 1b - - YES - - EG632971 139.439835 Entrez632971 ------LOC100043898 139.534026 Entrez100043898 ------Pik3c2g 139.570409 MGI:1203730 phosphatidylinositol 3-kinase, C2 domain containing, gamma polypeptide YES - YES - - Plcz1 139.936274 MGI:2150308 phospholipase C, zeta 1 YES - YES - - Aebp2 140.570928 MGI:1338038 AE binding protein 2 - - - - - LOC664875 141.086568 Entrez664875 ------EG435927 141.655526 MGI:3643685 predicted gene, EG435927 - - - - - Slco1a6 142.033435 MGI:1351906 solute carrier organic anion transporter family, member 1a6 - - - - - Sox5 143.779545 MGI:98367 SRY-box containing gene 5 - - - - - Bcat1 144.945689 MGI:104861 branched chain aminotransferase 1, cytosolic - - - - - Lrmp 145.068362 MGI:108424 lymphoid-restricted membrane protein - - - - - Casc1 145.121549 MGI:2444480 cancer susceptibility candidate 1 - - - - - Kras 145.163383 MGI:96680 v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog YES - - - - LOC100042866 145.212994 Entrez100042866 ------Ifltd1 145.343842 MGI:1921321 intermediate filament tail domain containing 1 - - - - - LOC100042879 145.489981 Entrez100042879 ------Itpr2 146.058019 MGI:99418 inositol 1,4,5-triphosphate receptor 2 YES - - - - 4933424B01Rik 146.496403 MGI:1918427 RIKEN cDNA 4933424B01 gene - - - - - Fgfr1op2 146.524536 MGI:1914779 FGFR1 oncogene partner 2 - - - - - Tm7sf3 146.548906 MGI:1914873 transmembrane 7 superfamily member 3 - - - - - Stk38l 146.672928 MGI:1922250 serine/threonine kinase 38 like - - - - - Arntl2 146.744587 MGI:2684845 aryl hydrocarbon receptor nuclear translocator-like 2 - - - - - Ppfibp1 146.835284 MGI:1914783 PTPRF interacting protein, binding protein 1 (liprin beta 1) - - - - - 2210417D09Rik 146.979113 MGI:1913782 RIKEN cDNA 2210417D09 gene - - - - - Mrps35 146.989349 MGI:2385255 mitochondrial ribosomal protein S35 - - - - - EG545893 147.021742 MGI:3645619 predicted gene, EG545893 - - - - - Klhdc5 147.037636 MGI:2444786 kelch domain containing 5 - - - - - Ccdc91 147.422459 MGI:1914265 coiled-coil domain containing 91 - YES - - - LOC100039712 147.589656 Entrez100039712 ------LOC100039727 147.624536 Entrez100039727 ------LOC100039736 147.656515 Entrez100039736 - - - YES - - Mlstd1 147.99452 MGI:2687035 male sterility domain containing 1 - - - - - Tmtc1 148.183217 MGI:3039590 transmembrane and tetratricopeptide repeat containing 1 - - - - -