Accelerated development of collapsing glomerulopathy in mice congenic for the HIVAN1 locus
2008; Elsevier BV; Volume: 75; Issue: 4 Linguagem: Inglês
10.1038/ki.2008.625
ISSN1523-1755
AutoresKa-Tak Chan, Natalia Papeta, Jeremiah Martino, Zongyu Zheng, Rachelle Z. Frankel, Paul E. Klotman, Vivette D. D’Agati, Richard P. Lifton, Ali G. Gharavi,
Tópico(s)Renal and related cancers
ResumoHIV-1 transgenic mice on the FVB/NJ background (TgFVB) are a well validated model of HIV-associated nephropathy (HIVAN). A mapping study between TgFVB and CAST/EiJ (CAST) strains showed this trait to be influenced by a major susceptibility locus on chromosome 3A1–A3 (HIVAN1), with CAST alleles associated with increased risk of disease. We introgressed a 50 Mb interval, encompassing this HIVAN1 locus, from CAST into the TgFVB genome (TgFVB-HIVAN1CAST congenic mice). Compared to the TgFVB strain, these congenic mice developed an earlier onset of proteinuria, a rapid progression to kidney failure, and increased mortality. A prospective study of these congenic mice also showed that they had a significantly greater histologic and biochemical evidence of glomerulopathy with one-third of mice developing global glomerulosclerosis by 6 weeks of age. An F2 cross between TgFVB and the congenic mice identified a significant linkage (LOD=3.7) to a 10 cM interval within the HIVAN1 region between D3Mit167 and D3Mit67 resulting in a 60% reduction of the original interval. These data independently confirm that a gene on chromosome 3A1-A3 increases susceptibility to HIVAN, resulting in early onset and rapid progression of kidney disease. These mice represent a new model to study the development and progression of collapsing glomerulopathy. HIV-1 transgenic mice on the FVB/NJ background (TgFVB) are a well validated model of HIV-associated nephropathy (HIVAN). A mapping study between TgFVB and CAST/EiJ (CAST) strains showed this trait to be influenced by a major susceptibility locus on chromosome 3A1–A3 (HIVAN1), with CAST alleles associated with increased risk of disease. We introgressed a 50 Mb interval, encompassing this HIVAN1 locus, from CAST into the TgFVB genome (TgFVB-HIVAN1CAST congenic mice). Compared to the TgFVB strain, these congenic mice developed an earlier onset of proteinuria, a rapid progression to kidney failure, and increased mortality. A prospective study of these congenic mice also showed that they had a significantly greater histologic and biochemical evidence of glomerulopathy with one-third of mice developing global glomerulosclerosis by 6 weeks of age. An F2 cross between TgFVB and the congenic mice identified a significant linkage (LOD=3.7) to a 10 cM interval within the HIVAN1 region between D3Mit167 and D3Mit67 resulting in a 60% reduction of the original interval. These data independently confirm that a gene on chromosome 3A1-A3 increases susceptibility to HIVAN, resulting in early onset and rapid progression of kidney disease. These mice represent a new model to study the development and progression of collapsing glomerulopathy. HIV-1 infection is associated with a number of pathologically distinct glomerulopathies that can lead to renal failure.1.Han T.M. Naicker S. Ramdial P.K. et al.A cross-sectional study of HIV-seropositive patients with varying degrees of proteinuria in South Africa.Kidney Int. 2006; 69: 2243-2250Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 2.Gerntholtz T.E. Goetsch S.J. Katz I. HIV-related nephropathy: a South African perspective.Kidney Int. 2006; 69: 1885-1891Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 3.Kiryluk K. Martino J. Gharavi A.G. Genetic susceptibility, HIV infection, and the kidney.Clin J Am Soc Nephrol. 2007; 2: S25-S35Crossref PubMed Scopus (19) Google Scholar, 4.D'Agati V.D. Podocyte injury in focal segmental glomerulosclerosis: lessons from animal models (a play in five acts).Kidney Int. 2008; 73: 399-406Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 5.D'Agati V. Appel G.B. HIV infection and the kidney.J Am Soc Nephrol. 1997; 8: 138-152PubMed Google Scholar, 6.Albaqumi M. Soos T.J. Barisoni L. et al.Collapsing glomerulopathy.J Am Soc Nephrol. 2006; 17: 2854-2863Crossref PubMed Scopus (113) Google Scholar Among these glomerulopathies, HIV-associated nephropathy (HIVAN) is the most common form and is characterized by the development of collapsing glomerulopathy and microcystic tubular dilatation.5.D'Agati V. Appel G.B. HIV infection and the kidney.J Am Soc Nephrol. 1997; 8: 138-152PubMed Google Scholar, 6.Albaqumi M. Soos T.J. Barisoni L. et al.Collapsing glomerulopathy.J Am Soc Nephrol. 2006; 17: 2854-2863Crossref PubMed Scopus (113) Google Scholar Most patients with HIVAN progress to end-stage renal failure without antiretroviral therapy, directly implicating HIV-1 in the pathogenesis of disease.4.D'Agati V.D. Podocyte injury in focal segmental glomerulosclerosis: lessons from animal models (a play in five acts).Kidney Int. 2008; 73: 399-406Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 7.Winston J.A. Bruggeman L.A. Ross M.D. et al.Nephropathy and establishment of a renal reservoir of HIV type 1 during primary infection.N Engl J Med. 2001; 344: 1979-1984Crossref PubMed Scopus (259) Google Scholar, 8.Bruggeman L.A. Ross M.D. Tanji N. et al.Renal epithelium is a previously unrecognized site of HIV-1 infection.J Am Soc Nephrol. 2000; 11: 2079-2087PubMed Google Scholar Data suggest that HIVAN is produced by HIV-1-induced dysregulation of glomerular podocytes, the highly differentiated visceral epithelial cells lining the glomerular filtration barrier.4.D'Agati V.D. Podocyte injury in focal segmental glomerulosclerosis: lessons from animal models (a play in five acts).Kidney Int. 2008; 73: 399-406Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar In HIVAN, podocytes exhibit enhanced proliferation and apoptosis and lose signature proteins of maturity such as podocin and synaptopodin.7.Winston J.A. Bruggeman L.A. Ross M.D. et al.Nephropathy and establishment of a renal reservoir of HIV type 1 during primary infection.N Engl J Med. 2001; 344: 1979-1984Crossref PubMed Scopus (259) Google Scholar, 9.Barisoni L. Bruggeman L.A. Mundel P. et al.HIV-1 induces renal epithelial dedifferentiation in a transgenic model of HIV-associated nephropathy.Kidney Int. 2000; 58: 173-181Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 10.He J.C. Husain M. Sunamoto M. et al.Nef stimulates proliferation of glomerular podocytes through activation of Src-dependent Stat3 and MAPK1,2 pathways.J Clin Invest. 2004; 114: 643-651Crossref PubMed Scopus (140) Google Scholar The mechanism underlying the dysregulated podocyte phenotype is not fully understood. HIV-1 has been shown to interfere with multiple pathways that are critical for maintaining cellular quiescence, with the HIV-1 nef protein as an important pathogenic mediator.10.He J.C. Husain M. Sunamoto M. et al.Nef stimulates proliferation of glomerular podocytes through activation of Src-dependent Stat3 and MAPK1,2 pathways.J Clin Invest. 2004; 114: 643-651Crossref PubMed Scopus (140) Google Scholar, 11.Martinka S. Bruggeman L.A. Persistent NF-kappaB activation in renal epithelial cells in a mouse model of HIV-associated nephropathy.Am J Physiol Renal Physiol. 2006; 290: F657-F665Crossref PubMed Scopus (35) Google Scholar, 12.Ross M.J. Martinka S. D'Agati V.D. et al.NF-kappaB regulates Fas-mediated apoptosis in HIV-associated nephropathy.J Am Soc Nephrol. 2005; 16: 2403-2411Crossref PubMed Scopus (54) Google Scholar, 13.Tandon R. Levental I. Huang C. et al.HIV infection changes glomerular podocyte cytoskeletal composition and results in distinct cellular mechanical properties.Am J Physiol Renal Physiol. 2007; 292: F701-F710Crossref PubMed Scopus (28) Google Scholar, 14.Lu T.C. He J.C. Wang Z.H. et al.HIV-1 Nef Disrupts the Podocyte Actin Cytoskeleton by Interacting with Diaphanous Interacting Protein.J Biol Chem. 2008; 283: 8173-8182Crossref PubMed Scopus (79) Google Scholar, 15.Sunamoto M. Husain M. He J.C. et al.Critical role for Nef in HIV-1-induced podocyte dedifferentiation.Kidney Int. 2003; 64: 1695-1701Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 16.Husain M. D'Agati V.D. He J.C. et al.HIV-1 Nef induces dedifferentiation of podocytes in vivo: a characteristic feature of HIVAN.AIDS. 2005; 19: 1975-1980Crossref PubMed Scopus (75) Google Scholar, 17.Zhong J. Zuo Y. Ma J. et al.Expression of HIV-1 genes in podocytes alone can lead to the full spectrum of HIV-1-associated nephropathy.Kidney Int. 2005; 68: 1048-1060Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 18.Zuo Y. Matsusaka T. Zhong J. et al.HIV-1 genes vpr and nef synergistically damage podocytes, leading to glomerulosclerosis.J Am Soc Nephrol. 2006; 17: 2832-2843Crossref PubMed Scopus (89) Google Scholar Host genetic factors also have an important impact on the course of HIV-1 infection and the development of complications.3.Kiryluk K. Martino J. Gharavi A.G. Genetic susceptibility, HIV infection, and the kidney.Clin J Am Soc Nephrol. 2007; 2: S25-S35Crossref PubMed Scopus (19) Google Scholar, 19.Smith M.W. Dean M. Carrington M. et al.Contrasting genetic influence of CCR2 and CCR5 variants on HIV-1 infection and disease progression. Hemophilia Growth and Development Study (HGDS), Multicenter AIDS Cohort Study (MACS), Multicenter Hemophilia Cohort Study (MHCS), San Francisco City Cohort (SFCC), ALIVE Study.Science. 1997; 277: 959-965Crossref PubMed Scopus (790) Google Scholar, 20.Gonzalez E. Kulkarni H. Bolivar H. et al.The influence of CCL3L1 gene-containing segmental duplications on HIV-1/AIDS susceptibility.Science. 2005; 307: 1434-1440Crossref PubMed Scopus (927) Google Scholar For example, the progression of HIV-1 disease is influenced by HLA genotype21.Moore C.B. John M. James I.R. et al.Evidence of HIV-1 adaptation to HLA-restricted immune responses at a population level.Science. 2002; 296: 1439-1443Crossref PubMed Scopus (628) Google Scholar or copy number variation at the CCL3L1 locus,20.Gonzalez E. Kulkarni H. Bolivar H. et al.The influence of CCL3L1 gene-containing segmental duplications on HIV-1/AIDS susceptibility.Science. 2005; 307: 1434-1440Crossref PubMed Scopus (927) Google Scholar and the development of dementia is influenced by APOE alleles.22.Corder E.H. Robertson K. Lannfelt L. et al.HIV-infected subjects with the E4 allele for APOE have excess dementia and peripheral neuropathy.Nat Med. 1998; 4: 1182-1184Crossref PubMed Scopus (202) Google Scholar Several lines of evidence also suggest that host genetic factors are also important for the development of HIVAN. Contrary to other glomerular diseases associated with HIV-1 infection, HIVAN almost exclusively occurs in individuals of African ancestry.1.Han T.M. Naicker S. Ramdial P.K. et al.A cross-sectional study of HIV-seropositive patients with varying degrees of proteinuria in South Africa.Kidney Int. 2006; 69: 2243-2250Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 2.Gerntholtz T.E. Goetsch S.J. Katz I. HIV-related nephropathy: a South African perspective.Kidney Int. 2006; 69: 1885-1891Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 23.US Renal Data System USRDS 2007 Annual Data Report: Atlas of End-Stage Renal Disease in the United States. National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD2007Google Scholar, 24.Winston J.A. Klotman M.E. Klotman P.E. HIV-associated nephropathy is a late, not early, manifestation of HIV-1 infection.Kidney Int. 1999; 55: 1036-1040Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar Moreover, family members of HIVAN patients also have a higher incidence of end-stage renal disease, suggesting segregation of nephropathy-predisposing alleles in these families.25.Freedman B.I. Soucie J.M. Stone S.M. et al.Familial clustering of end-stage renal disease in blacks with HIV- associated nephropathy.Am J Kidney Dis. 1999; 34: 254-258Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar Most recently, studies have shown that sequence variants in the podocyte-expressed non-muscle myosin heavy chain gene (MYH9) are associated with multiple nephropathies including HIVAN, explaining these epidemiological observations among African Americans.26.Kao W.H. Klag M.J. Meoni L.A. Family Investigation of Nephropathy and Diabetes Research Group et al.MYH9 is associated with nondiabetic end-stage renal disease in African Americans.Nat Genet. 2008; 40: 1185-1192Crossref PubMed Scopus (506) Google Scholar, 27.Kopp J.B. Smith M.W. Nelson G.W. et al.MYH9 is a major-effect risk gene for focal segmental glomerulosclerosis.Nat Genet. 2008; 40: 1175-1184Crossref PubMed Scopus (555) Google Scholar Multiple murine models of HIVAN have been developed and highlight the importance of permissive genetic background on the development of this trait. Introduction of the HIV-1 proviral genome, or specific HIV-1 genes (such as nef) into the mouse genome reproduces all the clinical and pathological features of HIVAN.15.Sunamoto M. Husain M. He J.C. et al.Critical role for Nef in HIV-1-induced podocyte dedifferentiation.Kidney Int. 2003; 64: 1695-1701Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 16.Husain M. D'Agati V.D. He J.C. et al.HIV-1 Nef induces dedifferentiation of podocytes in vivo: a characteristic feature of HIVAN.AIDS. 2005; 19: 1975-1980Crossref PubMed Scopus (75) Google Scholar, 17.Zhong J. Zuo Y. Ma J. et al.Expression of HIV-1 genes in podocytes alone can lead to the full spectrum of HIV-1-associated nephropathy.Kidney Int. 2005; 68: 1048-1060Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 18.Zuo Y. Matsusaka T. Zhong J. et al.HIV-1 genes vpr and nef synergistically damage podocytes, leading to glomerulosclerosis.J Am Soc Nephrol. 2006; 17: 2832-2843Crossref PubMed Scopus (89) Google Scholar, 28.Kopp J.B. Klotman M.E. Adler S.H. et al.Progressive glomerulosclerosis and enhanced renal accumulation of basement membrane components in mice transgenic for human immunodeficiency virus type 1 genes.Proc Natl Acad Sci USA. 1992; 89: 1577-1581Crossref PubMed Scopus (270) Google Scholar However, the development of disease is highly strain dependent. Studies using independent transgene constructs have shown that the FVB/NJ (FVB) genetic background is highly permissive, resulting in all the manifestations of collapsing glomerulopathy.17.Zhong J. Zuo Y. Ma J. et al.Expression of HIV-1 genes in podocytes alone can lead to the full spectrum of HIV-1-associated nephropathy.Kidney Int. 2005; 68: 1048-1060Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 18.Zuo Y. Matsusaka T. Zhong J. et al.HIV-1 genes vpr and nef synergistically damage podocytes, leading to glomerulosclerosis.J Am Soc Nephrol. 2006; 17: 2832-2843Crossref PubMed Scopus (89) Google Scholar, 28.Kopp J.B. Klotman M.E. Adler S.H. et al.Progressive glomerulosclerosis and enhanced renal accumulation of basement membrane components in mice transgenic for human immunodeficiency virus type 1 genes.Proc Natl Acad Sci USA. 1992; 89: 1577-1581Crossref PubMed Scopus (270) Google Scholar, 29.Gharavi A.G. Ahmad T. Wong R.D. et al.Mapping a locus for susceptibility to HIV-1-associated nephropathy to mouse chromosome 3.Proc Natl Acad Sci USA. 2004; 101: 2488-2493Crossref PubMed Scopus (84) Google Scholar To examine the impact of host genetic factors, we previously produced F1 hybrids between HIV-1 transgenic FVB lines (TgFVB) and six inbred strains and found wide variation in penetrance of disease.29.Gharavi A.G. Ahmad T. Wong R.D. et al.Mapping a locus for susceptibility to HIV-1-associated nephropathy to mouse chromosome 3.Proc Natl Acad Sci USA. 2004; 101: 2488-2493Crossref PubMed Scopus (84) Google Scholar Using a cross between TgFVB and the CAST/EiJ (CAST) strain, we identified a major susceptibility locus for HIVAN on chromosome 3A1–A3, which we named HIVAN1.29.Gharavi A.G. Ahmad T. Wong R.D. et al.Mapping a locus for susceptibility to HIV-1-associated nephropathy to mouse chromosome 3.Proc Natl Acad Sci USA. 2004; 101: 2488-2493Crossref PubMed Scopus (84) Google Scholar Interestingly, at the HIVAN1 locus, CAST alleles were associated with increased severity of disease. However, the HIVAN1 95th percentile confidence interval spanned over 50 Mb, necessitating additional experiment for identification of the underlying gene. Derivation of congenic lines trapping susceptibility loci is a powerful approach for replication of linkage findings and subsequent elucidation of complex traits.30.Abiola O. Angel J.M. Avner P. Complex Trait Consortium et al.The nature and identification of quantitative trait loci: a community's view.Nat Rev Genet. 2003; 4: 911-916PubMed Google Scholar Congenic lines are produced by introgressing chromosomal segments encompassing the quantitative trait loci (QTL) of interest from a donor strain into a recipient strain, thereby enabling analysis of alternative alleles of the QTL within the same genetic background.31.Darvasi A. Experimental strategies for the genetic dissection of complex traits in animal models.Nat Genet. 1998; 18: 19-24Crossref PubMed Scopus (443) Google Scholar This procedure provides independent verification of the initial linkage studies, enables better characterization of the phenotypic effect imparted by the QTL, and facilitates gene identification by fine mapping or derivation of subcongenic lines.30.Abiola O. Angel J.M. Avner P. Complex Trait Consortium et al.The nature and identification of quantitative trait loci: a community's view.Nat Rev Genet. 2003; 4: 911-916PubMed Google Scholar, 31.Darvasi A. Experimental strategies for the genetic dissection of complex traits in animal models.Nat Genet. 1998; 18: 19-24Crossref PubMed Scopus (443) Google Scholar Toward identification of the HIVAN1 gene, we produced mice congenic for the HIVAN1 interval and report genetic and phenotypic analysis of this new model of nephropathy. We produced HIVAN1 mice congenic (named the TgFVB-HIVAN1CAST strain) by introducing the chromosome segment encompassing this interval from CAST into the FVB genome by 10 generations of backcrossing. A genome scan with 82 informative loci in the 10th backcross generation confirmed that the TgFVB-HIVAN1CAST strain possessed a pure FVB genetic background outside the introgressed region. The proximal and distal loci homozygous for CAST alleles were delimited by rs6372626 (located at 4.2 Mb on the mouse reference sequence) and rs13477119 (54.6 Mb), respectively; the next proximal and distal markers homozygous for FVB alleles were rs6171250 (3 Mb) and rs30305237 (55.8 Mb), respectively. Thus the size of the congenic segment spans from a minimum of 50.4 Mb to a maximum of 52.8 Mb. Nontransgenic FVB-HIVAN1CAST mice were phenotypically normal with no evidence of nephropathy or proteinuria. However, HIV-1 transgenic counterparts developed rapid onset of kidney disease (Figure 1) with ∼40% mortality rate at 8 weeks of age (compared to <5% for TgFVB). Moreover, by 4 weeks of age, HIV-1 transgenic TgFVB-HIVAN1CAST mice displayed increased incidence of proliferative epidermal lesions characteristic of animals expressing this HIV-1-transgene construct,32.Kopp J.B. Rooney J.F. Wohlenberg C. et al.Cutaneous disorders and viral gene expression in HIV-1 transgenic mice.AIDS Res Hum Retroviruses. 1993; 9: 267-275Crossref PubMed Scopus (35) Google Scholar necessitating euthanasia based on humane criteria. Therefore, to prospectively study the impact of HIVAN1 alleles on the development of kidney disease, a cohort of TgFVB-HIVAN1CAST mice were studied from birth to 6 weeks of age and compared to TgFVB counterpart. At 3 weeks of age, all HIV-1 transgenic mice displayed significant proteinuria compared to nontransgenic mice, but homozygosity for CAST alleles was associated with a twofold increase in albuminuria (P<0.04; Figure 2a). At 6 weeks of age, TgFVB-HIVAN1CAST mice continued to display significantly greater proteinuria compared to TgFVB mice (P<0.02; Figure 2a). On histologic assessment, TgFVB-HIVAN1CAST demonstrated hallmarks of collapsing glomerulopathy, including tuft collapse and overlying podocyte proliferation and hypertrophy (Figure 1). Quantitative comparison of renal histopathology demonstrated significantly increased glomerulosclerosis (P=0.003; Figure 2b) and tubular injury with microcystic dilatation (P=0.002; Figure 2b) in TgFVB-HIVAN1CAST compared to TgFVB mice. Interestingly, 9/25 TgFVB-HIVAN1CAST animals also demonstrated global glomerulosclerosis with extensive podocyte depletion at 6 weeks of age, a finding that was not observed in their TgFVB counterparts (P=0.002 on Fisher's exact test). Heterozygous mice also displayed increased pathology, intermediate between FVB/FVB and CAST/CAST, consistent with an additive effect of CAST alleles (Figure 2a and b).Figure 2Phenotypic comparison of TgFVB and TgFVB-HIVAN1CAST mice. Comparison of proteinuria (a) and glomerulosclerosis scores (b) in HIV-1 transgenic mice carrying different alleles at the HIVAN1 locus on chromosome 3A1–A3. Values for a cohort of nontransgenic (nonTg) mice of all three genotypes are shown for comparisons. (c) Comparison of HIV-1 Env expression in mice carrying different alleles at the HIVAN1 locus. #P<0.005, *P<0.05 vs Tg FVB/FVB genotype.View Large Image Figure ViewerDownload (PPT) Previous studies in TgFVB mice have shown that the development of nephropathy coincides with increased expression of the HIV-1 transgene, followed by gradual reduction in expression secondary to loss of renal epithelial cells accompanying progressive renal injury.27.Kopp J.B. Smith M.W. Nelson G.W. et al.MYH9 is a major-effect risk gene for focal segmental glomerulosclerosis.Nat Genet. 2008; 40: 1175-1184Crossref PubMed Scopus (555) Google Scholar To assess whether increased severity of kidney disease in TgFVB-HIVAN1CAST mice is attributable to enhanced transgene expression, we quantitated transgene expression in 10 CAST/CAST and 10 FVB/FVB congenic mice with mild to moderate renal injury. These mice were matched for severity of renal histology scores to avoid bias introduced by secondary decrease in transgene expression due to nephropathy. There were no differences in transgene expression between the two genotype groups (Figure 2c), suggesting that increased severity of kidney disease is mediated through variation in host response to HIV-1 rather than enhanced expression of the transgene. These data demonstrate successful trapping of the HIVAN1 susceptibility alleles in our congenic strain, providing a novel tool for identifying the underlying susceptibility allele. We next utilized this strain to achieve further refinement of the HIVAN1 interval by meiotic mapping. We produced an F2 intercross between TgFVB and FVB-HIVAN1CAST strains and phenotyped 68 F2 progeny. Genotypic analysis with seven informative markers demonstrated significant linkage of renal histology score with the HIVAN1 locus (peak LOD 3.7, empiric P=0.001 and 3.3, empiric P=0.004, at D3Mit224 for glomerular and tubular injury scores, respectively; Figure 3a). As a single interval was tested, these LOD scores achieve pointwise significance for replication (P 75% of tissue affected. The protocol was approved by the IACUC committee at the Columbia University Medical Center. The F2 cohort was genotyped using seven informative microsatellite markers across the HIVAN1 locus (Figure 3). Multipoint analysis of linkage was performed using the R/QTL package.47.Broman K.W. Wu H. Sen S. et al.R/qtl: QTL mapping in experimental crosses.Bioinformatics. 2003; 19: 889-890Crossref PubMed Scopus (2047) Google Scholar As the renal injury scores were not normally distributed, a nonparametric model was used. In addition, we conducted 10,000 permutations of phenotypes on genotype to determine the empirical significance of the linkage findings. Statistical analyses were performed using SPSS. The primary endpoints were the comparison of proteinuria and histologic injury between TgFVB and FVB-HIVAN1CAST strains using two-sided t-test. P-values≤0.05 were considered significant. SNP ID and annotation across the HIVAN1 interval were obtained from the Mouse Phenome Database (http://aretha.jax.org/pub-cgi/phenome/mpdcgi?rtn=docs/home). Non-IBD segments were identified by filtering for polymorphic SNP between FVB/NJ and CAST/eiJ strains. Gene annotation was performed using the PANTHER database (http://www.pantherdb.org/). All the authors declared no competing interests. This work is supported by NIH 1PO1 DK56492-01. AGG is supported by the Columbia Center for Glomerular diseases and the Emerald Foundation. Supplementary Data Table S. Positional candidates within the 95th percent confidence interval in F2 cross. Table S1. Positional candidates with nonsynonymlous variants. Supplementary material is linked to the online version of the paper at http://www.nature.com/ki Download .pdf (.07 MB) Help with pdf files Supplementary Data
Referência(s)