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HIV-associated nephropathy in African Americans11The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. The publisher or recipient acknowledges right of the U.S. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article.

2003; Elsevier BV; Volume: 63; Linguagem: Inglês

10.1046/j.1523-1755.63.s83.10.x

1523-1755

Jeffrey B. Kopp, Cheryl A. Winkler,

Hepatitis C virus research

HIV-associated nephropathy in African Americans. Human immunodeficiency virus-1 (HIV-1) infection is associated with several glomerular syndromes, the most prevalent of which is HIV-associated focal segmental glomerulosclerosis (FSGS). At present, HIV-associated FSGS may account for up to 30% of patients in the United States entering end-stage renal disease (ESRD) as a consequence of FSGS. The mechanisms responsible for HIV-associated FSGS are not well defined, but evidence has been presented in favor of direct infection of renal parenchymal cells and toxicity of HIV-1 accessory proteins. HIV-associated FSGS has a striking predilection for patients of African descent. This likely has a genetic basis, although the gene or genes responsible have not yet been identified. One approach is to examine candidate genes for polymorphisms that are associated with disease. Another approach uses a genome-wide scan, relying upon linkage disequilibrium between DNA markers and the disease gene, to identify the causal gene or genes. African Americans are an admixed population, with genetic contributions from African, European, and Native American populations. In admixed populations, linkage disequilibrium between disease genes and marker genes can be exploited to identify disease genes, using an approach termed mapping by admixture linkage disequilibrium (MALD). HIV-associated nephropathy in African Americans. Human immunodeficiency virus-1 (HIV-1) infection is associated with several glomerular syndromes, the most prevalent of which is HIV-associated focal segmental glomerulosclerosis (FSGS). At present, HIV-associated FSGS may account for up to 30% of patients in the United States entering end-stage renal disease (ESRD) as a consequence of FSGS. The mechanisms responsible for HIV-associated FSGS are not well defined, but evidence has been presented in favor of direct infection of renal parenchymal cells and toxicity of HIV-1 accessory proteins. HIV-associated FSGS has a striking predilection for patients of African descent. This likely has a genetic basis, although the gene or genes responsible have not yet been identified. One approach is to examine candidate genes for polymorphisms that are associated with disease. Another approach uses a genome-wide scan, relying upon linkage disequilibrium between DNA markers and the disease gene, to identify the causal gene or genes. African Americans are an admixed population, with genetic contributions from African, European, and Native American populations. In admixed populations, linkage disequilibrium between disease genes and marker genes can be exploited to identify disease genes, using an approach termed mapping by admixture linkage disequilibrium (MALD). Human immunodeficiency virus-1 (HIV-1) infection is associated with several glomerular syndromes, as first recognized in the mid-1980s Table 1. Most common among European, Hispanic, and Asian patients are immune complex glomerulonephritis, with deposition of either IgA or multiple immunoglobulins1Praditpornsilpa K. Napathorn S. Yenrudi S. et al.Renal pathology and HIV infection in Thailand.Am J Kidney Dis. 1999; 33: 282-286Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar,2Cantor E.S. Kimmel P.L. Bosch J.P. Effect of race on expression of acquired immunodeficiency syndrome-associated nephropathy.Arch Intern Med. 1991; 151: 125-128Crossref PubMed Scopus (99) Google Scholar. Most common in patients of African descent is focal segmental glomerulosclerosis (FSGS), which affects approximately 10% of HIV-infected patients. The term HIV-associated nephropathy has been used in different ways, including describing all HIV-associated glomerular disease, end-stage renal disease (ESRD) patients in patients with HIV-1 infection, and biopsy-proven HIV-associated FSGS. Due to the potential for confusion, in this review the term HIV-associated FSGS will be used.Table 1HIV-associated glomerular diseaseCommon Focal segmental glomerulosclerosis Mesangial proliferative glomerulonephritis IgA nephropathyUncommon Thrombotic microangiopathy Membranoproliferative glomerulonephritis (typically associated with HCV co-infection) Membranous nephropathy Amyloid Open table in a new tab HIV-associated FSGS may present with a range of urine protein excretion, from low level proteinuria (<2 g/day) to severe nephrotic proteinuria. Nephrotic syndrome is relatively uncommon, as is hypertension, although either may occur. HIV-associated FSGS was initially noted to have propensity to progress to ESRD, often within six months. In the early days of the HIV-1 epidemic, the course of HIV-associated FSGS was noted to be unusually fast, often progressing to ESRD in months. In recent years, with the advent of highly active anti-retroviral therapy (HAART) and the widespread use of angiotensin antagonist therapy, the clinical picture has changed dramatically. With effective anti-retroviral treatment, the renal disease often stabilizes, reverses, and may even disappear3Winston 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 (260) Google Scholar. The number of patients in the United States Renal Data Systems (USRDS) database beginning ESRD due to AIDS nephropathy peaked in 1995 and had declined slightly since, the timing consistent with beneficial effects of HAART, which was introduced in 1996. The pathologic features of HIV-associated FSGS include segmental glomerulosclerosis or solidification. The glomerular capillary tuft may show global or segmental capillary collapse; the latter may be a subtle finding that is distinguished from solidification by the relatively scant amount of periodic acid Schiff (PAS)-stained matrix protein. The podocytes are hyperplastic and may have proliferated to form a pseudo-crescent. The tubular epithelium shows focal flattening, atrophy, and formation of microcysts. The interstitium frequently has edema and a mononuclear infiltrate that may be out of proportion to the severity of glomerular involvement. Ultrastructurally, glomerular endothelial cells may manifest tubuloreticular inclusions, in common with lupus nephritis and thought to represent the effects of cytokines (possibly interferon-α). The racial discrepancy in susceptibility to HIV-associated FSGS is quite striking, with patients of African descent being at substantially increased risk as reported in studies from the USA, France and Brazil4Lopes G.S. Marques L.P.J. Rioja L.S. et al.Glomerular disease and human immunodeficiency virus infection in Brazil.Am J Nephrol. 1992; 12: 281-287Crossref PubMed Scopus (40) Google Scholar,5Nochy D. Gloz D. Dosquet P. et al.Renal disease associated with HIV infection: A multicentric study of 60 patients from Paris hospitals.Nephrol Dial Transplant. 1993; 8: 11-19PubMed Google Scholar. Similar comparative data are not available from Africa. Nevertheless, it is notable that reports from Zaire indicate that FSGS became the dominant cause of the nephrotic syndrome in a study of renal biopsies performed between 1986 and 1989, displacing minimal change disease and amyloid, which had been the leading causes prior to the appearance of HIV-16Pakasa M. Mangani N. Dikassa L. Focal and segmental glomerulosclerosis in nephrotic syndrome: A new profile of adult nephrotic syndrome in Zaire.Mod Pathol. 1993; 6: 125-128PubMed Google Scholar. The USRDS database lists 849 patients who entered ESRD in 1999, the last year for which data are available, with a diagnosis of AIDS nephropathy (a renal biopsy is not required for this diagnosis). Of the 824 patients for whom race was known, 94% were African American and 6% were white. The CDC Division of HIV-AIDS Prevention indicates that as of December 1999, 412,471 individuals were alive with HIV-1 infection. As of December 1999, a cumulative total of 733,374 patients had been infected with HIV-1 and of these 38% were black, not Hispanic, and 43% were white, not Hispanic. From these data, we estimate that the annual risk for ESRD due to AIDS nephropathy among African American patients is approximately 4945 per million HIV-1 infected individuals, and among white patients is approximately 276 per million HIV-1 infected individuals per year. Thus, the relative risk for AIDS nephropathy is approximately 18-fold increased among African Americans. Assuming that all patients with AIDS nephropathy have FSGS, then approximately 800 patients develop ESRD due to HIV-associated FSGS each year in the US. The USRDS data base indicates that approximately 2000 patients each year enter ESRD due to non-HIV associated FSGS (although this is likely an underestimate, since many patients with FSGS likely enter ESRD without a renal biopsy). Therefore, nearly 30% of FSGS incident ESRD patients have HIV-1 infection as the cause. True population-based estimates of the prevalence of FSGS among African Americans with HIV-1 infection are not available, as large cohorts have not been followed with sufficient attention to renal disease. An autopsy study from Texas suggested that 12% of African American patients dying with HIV-1 infection have histologically-confirmed FSGS; this is the best available estimate of prevalence7Shahinian V. Rajaraman S. Borucki M. et al.Prevalence of HIV-associated nephropathy in autopsies of HIV-infected patients.Am J Kidney Dis. 2000; 35: 884-888Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar. Further, while the number of HIV deaths have been declining, the fraction of death certificates that mention HIV and also mention renal disease is increasing8Kopp J.B. Renal dysfunction in HIV-1 infected patients.Curr Infect Dis Rep. 2002; 4: 449-460Crossref PubMed Google Scholar. In 1999, 10% of individuals (14% of blacks dying with HIV and 6% of whites dying with HIV) had renal disease (mostly renal failure) as a co-morbid condition. This places renal disease second only to liver disease (11%) as an associated condition at the time of death in HIV-infected patients. Thus, HIV-infected patients are living longer and healthier lives, but the prevalence of renal disease at the time of death continues to rise. Several lentiviruses are associated with FSGS, including HIV-1 in human, simian immunodeficiency virus (SIV) in monkey, and feline immunodeficiency virus (FIV) in cats. The mechanisms by which lentiviruses induce glomerular injury eventuating the pathologic syndrome of FSGS is not well understood, but several major mechanisms can be considered. First, there are scant data supporting opportunistic infection as a cause of HIV-associated FSGS. Lo and colleagues suggested that Mycoplasma fermentans was associated with FSGS9Bauer F.A. Wear D.J. Angritt P. Lo S.C. Mycoplasma fermentans (incognitus strain) infection in the kidneys of patients with acquired immunodeficiency syndrome and associated nephropathy: A light microscopic, immunohistochemical, and ultrastructural study.Hum Pathol. 1991; 22: 63-69Abstract Full Text PDF PubMed Scopus (65) Google Scholar. We have presented data that SV40 may contribute to FSGS in both HIV-infected and uninfected patients10Li R.M. Branton M.H. Tanawattanacharoen S. et al.Molecular identification of SV40 infection in human subjects and possible association with kidney disease.J Am Soc Nephrol. 2002; 13: 2320-2330Crossref PubMed Scopus (122) Google Scholar. Second, HIV-1 likely infects lymphocytes and macrophages that enter the kidney and might release inflammatory lymphokines, or cytokines, which promote injury and fibrosis. This hypothesis has not been evaluated directly. Third, there is a growing body of evidence that HIV-1 may infect renal parenchymal cells and elicit a cytopathic effect, including proliferation or apoptosis. In vitro experiments have demonstrated low level productive infection of cultured tubular epithelial cells11Ray P.E. Liu X.H. Henry D. et al.Infection of human primary renal epithelial cells with HIV-1 from children with HIV-associated nephropathy.Kidney Int. 1998; 53: 1217-1229Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar. Some investigators, but not others, have reported the infection of mesangial cells12Green D.F. Resnick L. Bourgoignie J.J. HIV infects endothelial and mesangial but not epithelial cells.Kidney Int. 1992; 41: 956-960Abstract Full Text PDF PubMed Scopus (101) Google Scholar, 13Alpers C. McClure J. Burstein S.L. Human mesangial cells are resistant to productive infection by multiple strains of human immunodeficiency virus types 1 and 2.Am J Kidney Dis. 1992; 19: 126-130Abstract Full Text PDF PubMed Scopus (40) Google Scholar, 14Tokizawa S. Shimizu N. Hui-Yu L. et al.Infection of mesangial cells with HIV and SIV: identification of GPR1 as a coreceptor.Kidney Int. 2000; 58: 607-617Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar. Infection of human podocytes has not been described. At the electron microscopic level, HIV-1 virions have not been demonstrated within the kidney. On the other hand, HIV-1 virions are also difficult to demonstrate in lymphoid tissue or in tissue macrophages, although they are readily demonstrated in cultured lymphocytes infected experimentally. Thus, the failure to demonstrate virions in the kidney may simply mean that infection occurs infrequently and is associated with low-level viral replication. Localization of HIV-1 protein and RNA to renal parenchymal cells has been controversial. While Cohen et al found both protein and RNA in podocytes and tubular epithelial cells15Cohen A.H. Sun N.C.J. Shapshak P. Imagawa D.T. Demonstration of human immunodeficiency virus in renal epithelium in HIV-associated nephropathy.Mod Pathol. 1989; 2: 125-128PubMed Google Scholar, others were unable to replicate these findings. Recently, Klotman and collaborators have developed sensitive in situ hybridization techniques and confirmed the initial localization of viral RNA and proviral DNA to podocyte and tubular epithelial cells16Bruggeman 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. Further, they have shown that podocytes have particular variants, suggesting that renal infection may involve selection for certain genotypes, and that renal epithelial cells may constitute a reservoir in patients receiving anti-retroviral treatment17Marras D. Bruggeman L.A. Gao F. et al.Replication and compartmentalization of HIV-1 in kidney epithelium of patients with HIV-associated nephropathy.Nat Med. 2002; 8: 522-526Crossref PubMed Scopus (246) Google Scholar. A fourth pathomechanism involves toxicity of HIV-1 proteins. Experiments using transgenic mice have demonstrated that particular HIV-1 accessory proteins can induce FSGS and interstitial nephritis Table 2, including the features characteristic of HIV-associated FSGS, such as focal and segmental capillary collapse and solidification of the glomerular capillary tuft, podocyte hyperplasia (but not tubulo-reticular inclusions in glomerular endothelial cells), microcystic tubular dilation, tubular atrophy, mononuclear interstitial infiltrate, proteinuria, and progressive renal dysfunction culminating in ESRD. These transgenic mice do not produce virions, since the gag and pol genes are lacking from the transgenes and, therefore, in this model system a replicating virus is not required to induce FSGS. Instead, these data point to a direct toxic effect of HIV proteins on renal cells. Two lines of evidence suggest that local renal production of these proteins is critical: lines that do not express the transgene in kidney but express the transgene in other tissues do not develop renal disease18Dickie P. Felser M. Eckhaus M. et al.HIV-associated nephropathy in transgenic mice expressing HIV-1 genes.Virology. 1991; 185: 109-119Crossref PubMed Scopus (227) Google Scholar, and renal disease follows the transgenic kidney in renal transplant experiments21Bruggeman L.A. Dikman S. Meng C. et al.Nephropathy in human immundeficiency virus-1 transgenic mice is due to renal transgene expression.J Clin Invest. 1997; 100: 84-92Crossref PubMed Scopus (217) Google Scholar.Table 2HIV-transgenic mice with renal renal diseaseReferencePromoterHIV genesRenal phenotypeDickie et al18Dickie P. Felser M. Eckhaus M. et al.HIV-associated nephropathy in transgenic mice expressing HIV-1 genes.Virology. 1991; 185: 109-119Crossref PubMed Scopus (227) Google ScholarLTRenv, tat, rev, nef, vif, vpr, vpuFSGSKimmel et al, (abstract)a(abstract; Kimmel et al, J Invest Med 44:321A, 1996)LTRtat, rev, nef, vif, vpr, vpuFSGSKajiyama et al19Kajiyama W. Klotman P.E. Dickie P. Kopp J.B. HIV-1 genes are expressed in glomerular and tubular epithelial cells in HIV-transgenic mouse kidney.AIDS Res Hum Retrovir. 1995; 11: S153Google ScholarLTRenv, tat, rev, vif, vpr, vpuFSGSJolicoeur et al20Hanna Z. Kay D.G. Rebai N. et al.Nef harbors a major determinant of pathogenicity for an AIDS-like disease induced by HIV-1 in transgenic mice.Cell. 1998; 95: 163-175Abstract Full Text Full Text PDF PubMed Scopus (404) Google ScholarCD4nefInterstitial nephritisDickie et al (unpublished observations)LTRtat, vprFSGSa (abstract; Kimmel et al, J Invest Med 44:321A, 1996) Open table in a new tab A provisional summary of the available data from the transgenic mice suggests that Tat or Vpr or both may induce FSGS, and that Nef may contribute to interstitial nephritis (without being strictly required). These viral proteins have complex effects on cell function and how these might induce renal injury is an exciting area for future work: Tat is a transactivating protein that increases production of viral RNA. Vpr (viral protein R) has multiple effects on host cells including G2 cell cycle arrest, regulation of apoptosis and cytokine production, and acting as both a transcriptional co-activator and co-repressor. Nef (originally named as a negative factor for viral replication) functions to down-regulate CD4 expression. Recently, Tat has been shown to induce proliferation in cultured podocytes, possibly by stimulating release of basic fibroblast growth factor22Conaldi P.G. Bottelli A. Baj A. et al.Human immunodeficiency virus-1 tat induces hyperproliferation and dysregulation of renal glomerular epithelial cells.Am J Pathol. 2002; 161: 53-61Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar. In summary, in mice viral accessory proteins induce many of the characteristic functional and histologic changes of HIV-associated FSGS. An important future step will be to extend these observations to human patients and to translate these insights into improved therapies for human patients. The transcription of HIV-1 viral RNA to proviral DNA by viral reverse transcriptase and then to viral RNA by host RNA polymerase II is relatively error prone, producing approximately 1 error for every 10,000 bp or one error per genome. Consequently, most virions are defective, but this genomic plasticity facilitates rapid viral evolution that enables the virus to evade host immunologic defenses and overcome antiviral therapy. A large number of viral quasi-species have been partially or fully sequenced. The env (envelope) gene encodes the transmembrane glycoprotein gp41 and the associated glycoprotein gp120. Env gp120 first binds CD4, which induces a conformational change in gp120 facilitating binding to one of several chemokine co-receptor molecules. At least six chemokine co-receptors are known, and in certain situations these may not require the presence of CD4. Variations in the sequence of gp120 account for the ability of particular HIV-1 variants to infect lymphocyte cell lines and some primary lymphocytes by using the chemokine receptor CXCR4 or macrophages and primary lymphocytes using CCR5. The receptors that are responsible for viral entry into renal parenchymal cells have not been well characterized. CD4 has not been convincingly demonstrated on any renal parenchymal cell in vivo, although RNA has been reported in cultured renal tubular epithelial cells. Mesangial cell infection has been proposed to be dependent on GPR114Tokizawa S. Shimizu N. Hui-Yu L. et al.Infection of mesangial cells with HIV and SIV: identification of GPR1 as a coreceptor.Kidney Int. 2000; 58: 607-617Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar. It could be hypothesized that particular viral variants have mutations in gp120 that either facilitate entry into renal cells, or that mutations in other viral proteins have particular toxicity for renal cells, but few data have been published addressing this issue. As noted earlier in this article, subjects of African descent are at markedly increased risk for FSGS in the setting of HIV-1 infection, with perhaps a 14-fold increased risk compared to Caucasians. Since a similar predilection is seen in studies from the US, France, and Brazil, cultural factors seem an unlikely explanation. Instead, it appears highly likely that one or more host genetic loci contributes, with particular risk alleles being more common in patients of African descent. These alleles might contribute to entry of HIV-1 into renal cells, might potentiate the toxicity of HIV-1 accessory proteins in renal cells, or might heighten a fibrotic response to HIV-1 initiated renal injury. The genetic mutation that contributes to HIV-associated FSGS might possibly (1) be unique for HIV-associated FSGS or (2) extend to idiopathic FSGS or (3) extend to other forms of glomerular injury. There is no evidence that the first possibility is true, but the relative rarity of HIV-1 infection (<0.2% of the US population is known to be infected) makes it difficult to estimate the risk for HIV-associated FSGS among different kindreds. On the other hand, it has been reported that probands with HIV-associated nephropathy have an increased number of first and second degree relatives with ESRD (but not necessarily FSGS) compared to ethnically-matched relatives with HIV-1 infection but without kidney disease23Freedman B.I. Soucie J.M. Stone S.M. Pegram S. 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. These data tend to support the second and especially the third possibility noted above. Thus, HIV-associated FSGS likely has a genetic component (or components) and an environmental component (viral infection), so that patients who do not develop HIV-1 infection are at increased risk for other forms of glomerular injury. What strategies are available for identifying genetic loci associated with complex diseases that may be both polygenic and have an environmental component? Approaches using classic family studies are ideal for simple Mendelian disease phenotypes, particularly those having high heritability and penetrance. However, family-based linkage studies have a low chance of success with low penetrant, multifactorial diseases. This is due both to the low number of affected individuals available in any kindred and the likelihood of mis-assigning unaffected individuals who in fact have the high-risk alleles but have not encountered the appropriate environmental stimulus. In situations where family-based linkage studies or sib-pair analyses are not feasible for the identification of disease genes, population-based studies using a genome-scan (indirect) or a candidate gene (direct) approach provide viable alternative strategies. The detection of polymorphic genes influencing common or low penetrant diseases such as non-familial FSGS is the goal of population-based association studies24Reich D.E. Lander E.S. On the allelic spectrum of human disease.Trends Genet. 2001; 17: 502-510Abstract Full Text Full Text PDF PubMed Scopus (863) Google Scholar, 25Cargill M. Altshuler D. Ireland J. et al.Characterization of single-nucleotide polymorphisms in coding regions of human genes.Nat Genet. 1999; 22: 231-238Crossref PubMed Scopus (1521) Google Scholar, 26Lander E.S. Schork N.J. Genetic dissection of complex traits.Science. 1994; 265: 2037-2048Crossref PubMed Scopus (2678) Google Scholar. Population-based association studies use distortions in allele or genotype frequencies between disease and control groups to detect disease genes, either by direct investigation of candidate genes or indirectly through investigation of genetic markers that track the causative gene or genes27Risch N.J. Searching for genetic determinants in the new millennium.Nature. 2000; 405: 847-856Crossref PubMed Scopus (1533) Google Scholar. For the direct gene approach, the selection of candidate genes should be based on biological insight that there may be a causal relationship between the genetic factor and the disease. However, this approach will miss unknown or unidentified causative genes. The indirect approach takes advantage of the nonrandom association (termed linkage disequilibrium) that exists between alleles on the same ancestral chromosomal segment. By carrying out a whole-genome scan, that is, genotyping DNA markers spaced at intervals across the human genome, it is possible to detect associations between the marker and the disease phenotype if the genetic marker is in linkage disequilibrium with the causative genetic factor28Devlin B. Risch N. A comparison of linkage disequilibrium measures for fine-scale mapping.Genomics. 1995; 29: 311-322Crossref PubMed Scopus (843) Google Scholar. This method has the significant advantage of identifying genes that were not previously identified as likely candidates. Identifying previously unsuspected genetic factors is likely to reveal unexpected mechanisms in disease pathogenesis. Candidate gene association studies compare the frequency of genetic polymorphisms for particular candidate genes between disease and control populations. Such studies, while commonly employed, have several potential pitfalls. First, the list of attractive candidate genes is often quite long. Second, markers within or near the candidate gene may not have been discovered, necessitating a lengthy search for polymorphisms in regulatory and coding regions of the candidate genes. Third, multiple comparisons may lead to spurious associations due to statistical fluctuations. Fourth, the choice of control populations is critical, as the disease and control populations may have differences in substructure (subtle differences in genetic background) that confound the result and may lead to non-causal associations. Population substructure can be minimized by careful matching of cases and controls for ethnicity and race, but the theoretical possibility of substructure within an ethnic/racial group remains. Fifth, even after a positive association is discovered within a candidate gene, the possibility remains that the association may result from linkage disequilibrium between the candidate gene and the true causal gene it is tracking. If the causal gene and its marker are in complete linkage disequilibrium, it may be impossible to determine the causal factor by genetic analysis alone. Criteria for validating genetic associations include the following: (1) replication in different study populations, (2) low statistical P values after correction for multiple comparisons, (3) high relative hazard or risk, and (4) functional data relating the genetic polymorphism to gene function and biological relevance with the disease29O'Brien S.J. Nelson G.W. Winkler C.A. Smith M.W. Polygenic and multifactorial disease gene association in man: Lessons from AIDS.Annu Rev Genet. 2000; 34: 563-591Crossref PubMed Scopus (65) Google Scholar. We have initiated studies comparing genetic polymorphisms among the following groups: African Americans with HIV-associated FSGS, African Americans with idiopathic FSGS, African Americans with HIV-1 infection but without renal disease (a hyper-normal control group, in that these individuals have been challenged by a virus known to induce FSGS but have not developed the syndrome), African American blood donors, European Americans with idiopathic FSGS, and European American blood donors. We are in the process of genotyping these subjects for a variety of candidate genes, including those for chemokine receptors, chemokines, cytokines, and members of the renin-angiotensin-transforming growth factor-β (TGF-β) system. To date, the only gene to show differences between African American patients with HIV-associated FSGS and African American control subjects is the angiotensin converting enzyme (ACE1) gene. Patients homozygous for the insertion allele in intron 16 are at increased risk for FSGS; this is true for African Americans with both HIV-associated FSGS and idiopathic FSGS (abstract; Kopp et al, J Am Soc Nephrol 9:390A, 1998). Analysis is underway for European American subjects, but the insertion allele is common in Caucasian populations as well, and so while it is unlikely that the ACE1 insertion/deletion is itself causal, it may be tracking the another polymorphic site elsewhere in the ACE1 gene. Linkage disequilibrium mapping is an indirect method of identifying causal disease genes based on linkage disequilibrium between a genetic marker and an adjacent disease gene27Risch N.J. Searching for genetic determinants in the new millennium.Nature. 2000; 405: 847-856Crossref PubMed Scopus (1533) Google Scholar,28Devlin B. Risch N. A comparison of linkage disequilibrium measures for fine-scale mapping.Genomics. 1995; 29: 311-322Crossref PubMed Scopus (843) Google Scholar. When a disease gene mutation occurs, it is in complete linkage disequilibrium with all flanking alleles. The rate of linkage disequilibrium decay to equilibrium is a function of the distance between the markers and elapsed time from the mutation event. Several processes tend to maintain or reintroduce linkage disequilibrium in human populations. These processes include recent admixture between two racial groups or populations (for example, admixture between Africans and Europeans), the founding of a new population by a small number of individuals (such as, the Polynesian explorers settling Hawaii), population bottle necks in which only a small number of individuals survive to reproduce (such as, population contraction following a lethal epidemic), and evolutionary selection (for example, selection pressure for an allele of one gene would influence the transmission of linked gene segments). In most white populations, the extent of linkage disequilibrium is limited to blocks of linkage disequilibrium of approximately 60 to 100 kb separated by small regions of high recombination (recombination is the exchange of genomic segments between sister chromosomes at each meiosis)30Goldstein D.B. Islands of linkage disequilibrium.Nat Genet. 2001; 29: 109-111Crossref PubMed Scopus (225) Google Scholar,31Reich D.E. Cargill M. Bolk S. et al.Linkage disequilibrium in the human genome.Nature. 2001; 411: 199-204Crossref PubMed Scopus (1289) Google Scholar. The number of genetic markers required to map by linkage disequilibrium is therefore daunting, somewhere on the order of 30,000 to 50,000 SNPs would be required for 0.1 centiMorgan (cM) (≅100 kb) coverage. Very recently, haplotype mapping has suggested that the haplotype blocks are much larger and more stable than previously believed. Knowledge of haplotype structure among populations will facilitate the selection of haplotype-specific SNP markers, thus increasing the resolution and power to identify disease genes32Gabriel S.B. Schaffer S.F. Nguyen H. et al.The structure of haplotype blocks in the human genome.Science. 2002; 296: 2225-2229Crossref PubMed Scopus (4532) Google Scholar. In Native Africans, the extent of linkage disequilibrium is much less since the populations are older with more recombination events, and therefore mapping by linkage disequilibrium would be even more difficult33Tishkoff S.A. Varkonyi R. Cahinhinan N. et al.Haplotype diversity and linkage disequilibrium at human G6PD: Recent origin of alleles that confer malarial resistance.Science. 2001; 293: 455-462Crossref PubMed Scopus (446) Google Scholar,34Briscoe D. Stephens J.C. O'Brien S.J. Linkage disequilibrium in admixed populations: Applications in gene mapping.J Hered. 1994; 85: 59-63PubMed Google Scholar. However, in recently admixed populations (such as African Americans or Hispanic Americans), both simulation and empirical studies have shown that linkage disequilibrium can extend over many cM before it is reshuffled by recombination34Briscoe D. Stephens J.C. O'Brien S.J. Linkage disequilibrium in admixed populations: Applications in gene mapping.J Hered. 1994; 85: 59-63PubMed Google Scholar,35McKeigue P.M. Carpenter J.R. Parra E.J. Shriver M.D. Estimation of admixture and detection of linkage in admixed populations by a Bayesian approach: Application to African-American populations.Ann Hum Genet. 2000; 64: 171-186Crossref PubMed Google Scholar. Theoretical calculations suggest that sufficient linkage for mapping studies remains for 10 to 20 generations after admixture began34Briscoe D. Stephens J.C. O'Brien S.J. Linkage disequilibrium in admixed populations: Applications in gene mapping.J Hered. 1994; 85: 59-63PubMed Google Scholar. If the ancestral populations have different genetic risks for a particular disease, linkage disequilibrium can be exploited to map the genes that underlie these risk differentials. The identification of genetic loci in admixed population has been termed mapping by admixture linkage disequilibrium (MALD). In practical terms, successful MALD requires a sufficiently large number of marker genes with allele frequencies that differ sufficiently between the ancestral populations and are distributed at approximately equal intervals across the genome36Smith M.W. Lautenberger J.A. Shin H.D. et al.Markers for mapping by admixture linkage disequilibrium in African American and Hispanic populations.Am J Hum Genet. 2001; 69: 1080-1094Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar. Data analysis involves comparing the prevalence of marker genes polymorphisms among the disease and control groups, looking for distortions that identify a particular genomic region in which there is a tendency for the disease population to inherit genes disproportionately from one ancestral population. Several approaches to testing for linkage disequilibrium have been proposed, including transmission disequilibrium testing and a Bayesian approach involving conditioning on parental admixture37Zheng C. Elston R.C. Multipoint linkage disequilibrium mapping with particular reference to the African-American population.Genet Epidemiol. 1999; 17: 79-101Crossref PubMed Scopus (34) Google Scholar,38McKeigue P.M. Mapping genes that underlie ethnic differences in disease risk: Methods for detecting linkage in admixed populations, by conditioning on parental admixture.Am J Hum Genet. 1998; 63: 241-251Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar. In the present example, this would mean a genomic segment that is disproportionately African in origin in African American patients with HIV-associated FSGS compared to control African American subjects. This approach is still in its infancy, and has not yet been successfully used to identify a disease gene. Patients of African descent in the New World represent an admixed population, comprised of genetic contributions from the ancestral populations in Africa, Europe and Asia (via Native Americans). It is estimated that 15 to 20% of the genes in African Americans in the US derive from the European population35McKeigue P.M. Carpenter J.R. Parra E.J. Shriver M.D. Estimation of admixture and detection of linkage in admixed populations by a Bayesian approach: Application to African-American populations.Ann Hum Genet. 2000; 64: 171-186Crossref PubMed Google Scholar. The Atlantic slave trade was responsible for bringing an estimated 12 million individuals to the Americas between 1450 and 1900, with the peak coming in the eighteenth century when over 6 million Africans made the Middle Passage39Iliffe J. Africans: The History of a Continent. Cambridge University Press, Cambridge1995Google Scholar,40Davidson B. The African Slave Trade. Little Brown, Boston1980Google Scholar. Mortality was appalling, with an average mortality of approximately 10 to 15% on the Middle Passage itself and a total mortality between capture inland and arrival in the Americas of approximately 50%. Genetic admixture between Africans, Europeans, and Native Americans may have began soon after arrival of the Africans in the New World, and has continued to varying degrees to the present time. Thus, the upper bound for the average duration of population admixture is approximately 250 to 300 years or perhaps 10 to 15 generations. In a test study, McKeigue and colleagues typed African Americans at 10 marker loci and found linkage between two marker loci located 22 cM apart. These data suggested European gene flow into the African American population began between five and nine generations ago34Briscoe D. Stephens J.C. O'Brien S.J. Linkage disequilibrium in admixed populations: Applications in gene mapping.J Hered. 1994; 85: 59-63PubMed Google Scholar. Thus, it appears that the US African American population is a suitable population to locate disease genes using MALD. By taking advantage of the persistent linkage disequilibrium that exists as a result of the admixture, linkage disequilibrium mapping may be achieved at a genome-wide coverage of only 5 to 10 cM and requiring only 300 to 600 markers. HIV-associated FSGS, with a marked increased risk among African Americans compared to Caucasians, is an attractive disease target for this approach. HIV-associated FSGS is a common complication of HIV-1 infection and is frequently responsive to effective anti-retroviral therapy. Likely pathomechanisms include direct infection of renal parenchymal cells and/or the toxicity of viral accessory proteins. HIV-associated FSGS has a particular predilection for patients of African descent. The genetic locus or loci responsible for this susceptibility have yet to be identified, but MALD is an attractive methodology to use in the search. It is likely that a better understanding of the pathogenesis of HIV-associated FSGS, including host genetic factors, will lead to more effective therapy.