African Americans are disproportionately affected by chronic and end stage renal disease (ESRD);while 35% of patients on dialysis are African American, only 13.2% of the U.S. population is African American.A major factor contributing to this disparity are genetic variations in apolipoprotein L1 (APOL1). APOL1 is aplasma protein protective against "African sleeping sickness' caused by the parasite Trypanosoma brucei. Thereare three main allelic variants of APOL1: G0 (wild-type), G1, and G2. The G1 and G2 APOL1 alleles (i.e., renalrisk alleles) impart resistance to sleeping sickness, while the G0 allele enables parasite survival and infection.For this reason, the G1 and G2 alleles are highly prevalent in individuals with African ancestry. The G1 and G2variants of APOL1 are also present at relatively high frequencies among African Americans, with approximately35% of the African American population having at least one G1 or G2 allele. Despite providing an advantage insurvival from African trypanosomiasis, these genotypic variants predispose individuals to develop severe,irreparable kidney disease. People with two risk alleles, i.e., who are homozygous for either the G1 or G2 allelesor are doubly heterozygous for these alleles (G1/G2), have an APOL1 "risk genotype' and are at elevated riskfor developing focal segmental glomerulosclerosis (FSGS), which leads to progressive scarring and loss offunction of glomeruli. Moreover, the risk genotype is associated with reduced allograft longevity in kidneystransplanted from donors with two risk alleles. More recently, it has been found that other glomerulopathies linkedto viral infections, including HIV and SARS-CoV-2, are exacerbated by having the APOL1 risk genotype. Giventhat approximately 13% of African Americans have a genotype with two risk alleles, APOL1-linked kidney diseaserepresents a potentially massive, yet still underappreciated, public health issue. The available methods fordetecting pathological APOL1 variants, including gene sequencing and TaqMan, are relatively expensive andrequire specialized equipment and skills. Recently, CRISPR/Cas-based methods of detecting specific nucleicacid sequences have been developed. These methods are both simple and inexpensive and therefore offersignificant advantages to conventional genotyping methods. In this Phase I application, we propose to developa proof-of-concept CRISPR/Cas12a-based genotyping assay to detect the G0, G1, and G2 variants of ApoL1.Once developed and optimized, this assay will lead to a suite of reagents and techniques to expand access tosimple and affordable ApoL1 genotyping that is less reliant on specialized equipment. Two novel therapeuticagents for treatment of APOL1-mediated kidney disease are currently in clinical trials, highlighting the urgencyto develop better diagnostic tools that can identify individuals who could benefit from these treatments.
Public Health Relevance Statement: PROJECT NARRATIVE. Approximately 13% of African Americans have a combination of pathological variants of the APOL1 gene, often termed the APOL1 "risk genotype," that predisposes individuals to serious kidney diseases such as focal segmental glomerulosclerosis (FSGS). Despite the potential severity of APOL1-linked kidney disease, genetic testing for APOL1 variants is uncommonly performed, primarily due to the expense and lack of availability of testing. In this application, we propose to develop a CRISPR/Cas12a-mediated genotyping platform capable of rapid and inexpensive detection of pathological APOL1 variants from patient samples.
Project Terms: Affect ; Alleles ; Allelomorphs ; Apolipoproteins ; Base Sequence ; Nucleotide Sequence ; nucleic acid sequence ; Biological Assay ; Assay ; Bioassay ; Biologic Assays ; Blood Circulation ; Bloodstream ; Circulation ; Cells ; Cell Body ; Cicatrix ; Scars ; Clinical Trials ; Dialysis procedure ; Dialysis ; dialysis therapy ; Donor person ; transplant donor ; Double-Blind Method ; Double-Blind Study ; Double-Blinded ; Double-Masked Method ; Double-Masked Study ; Equipment ; Exhibits ; Genes ; Genotype ; Health ; HIV ; AIDS Virus ; Acquired Immune Deficiency Syndrome Virus ; Acquired Immunodeficiency Syndrome Virus ; Human Immunodeficiency Viruses ; LAV-HTLV-III ; Lymphadenopathy-Associated Virus ; Virus-HIV ; Human ; Modern Man ; Incidence ; Infection ; Kidney ; Kidney Urinary System ; renal ; Kidney Diseases ; Nephropathy ; Renal Disease ; kidney disorder ; renal disorder ; Chronic Kidney Failure ; Chronic Renal Disease ; Chronic Renal Failure ; chronic kidney disease ; Kidney Glomerulus ; Malpighian Tuft ; kidney glomeruli ; renal glomerulus ; Kidney Transplantation ; Kidney Grafting ; Kidney Transplants ; Renal Grafting ; Renal Transplantation ; Renal Transplants ; kidney tx ; Laboratories ; High Density Lipoproteins ; HDL ; HDL Lipoproteins ; Heavy Lipoproteins ; High density lipoprotein ; alpha-Lipoproteins ; Longevity ; Length of Life ; life span ; lifespan ; Membrane Glycoproteins ; Cell Surface Glycoproteins ; Surface Glycoproteins ; Methods ; Parasites ; Patients ; Plasma Proteins ; pressure ; Proteins ; Public Health ; Reagent ; Risk ; Sensitivity and Specificity ; Specificity ; Testing ; Time ; Trypanosoma ; Trypanosome ; Trypanosoma brucei brucei ; T brucei ; T. brucei ; Trypanosoma brucei ; African Trypanosomiasis ; African Sleeping Sickness ; sleeping sickness ; United States ; Genetic Variation ; Genetic Diversity ; Virus Diseases ; Viral Diseases ; viral infection ; virus infection ; virus-induced disease ; Virus ; AIDS-Associated Nephropathy ; AIDS Nephropathy ; AIDS-Related Nephropathy ; HIV-Associated Nephropathy ; HIV-Related Nephropathy ; Human Immunodeficiency Virus-Associated Nephropathy ; African American ; Afro American ; Afroamerican ; Black Populations ; black American ; Mediating ; base ; Chronic ; Clinical ; Phase ; Variant ; Variation ; Link ; Serum ; Blood Serum ; Individual ; African ; Binding Proteins ; Ligand Binding Protein ; Ligand Binding Protein Gene ; Protein Binding ; bound protein ; Therapeutic Agents ; Double-Stranded DNA ; dsDNA ; ds-DNA ; tool ; Diagnostic ; Life ; FSGS ; Focal segmental glomerular sclerosis ; Focal Segmental Glomerulosclerosis ; Frequencies ; Severities ; Complex ; Techniques ; Allografting ; experience ; nuclease ; Accuracy of Diagnosis ; diagnostic accuracy ; skills ; Reporting ; Sampling ; Molecular Interaction ; Binding ; ESRD ; End-Stage Kidney Disease ; End-Stage Renal Disease ; End stage renal failure ; Detection ; Pathologic ; Molecular ; Development ; developmental ; point of care ; cost ; genetic variant ; Gene variant ; allele variant ; allelic variant ; genomic variant ; Population ; Prevalence ; Resistance ; resistant ; C-terminal ; novel therapeutics ; new drug treatments ; new drugs ; new therapeutics ; new therapy ; next generation therapeutics ; novel drug treatments ; novel drugs ; novel therapy ; loss of function ; functional loss ; high risk ; risk variant ; Risk-associated variant ; risk allele ; risk gene ; risk genotype ; risk loci ; risk locus ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR ; CRISPR/Cas system ; Genomic DNA ; gDNA ; APOL1 gene ; APOL-I ; APOL1 ; Innate Immune System ; DNA sequencing ; DNA seq ; DNAseq ; renal damage ; damage to kidney ; kidney damage ; COVID-19 ; COVID19 ; CV-19 ; CV19 ; corona virus disease 2019 ; coronavirus disease 2019 ; 2019-nCoV ; 2019 novel corona virus ; 2019 novel coronavirus ; COVID-19 virus ; COVID19 virus ; CoV-2 ; CoV2 ; SARS corona virus 2 ; SARS-CoV-2 ; SARS-CoV2 ; SARS-associated corona virus 2 ; SARS-associated coronavirus 2 ; SARS-coronavirus-2 ; SARS-related corona virus 2 ; SARS-related coronavirus 2 ; SARSCoV2 ; Severe Acute Respiratory Distress Syndrome CoV 2 ; Severe Acute Respiratory Distress Syndrome Corona Virus 2 ; Severe Acute Respiratory Distress Syndrome Coronavirus 2 ; Severe Acute Respiratory Syndrome CoV 2 ; Severe Acute Respiratory Syndrome-associated coronavirus 2 ; Severe Acute Respiratory Syndrome-related coronavirus 2 ; Severe acute respiratory syndrome associated corona virus 2 ; Severe acute respiratory syndrome corona virus 2 ; Severe acute respiratory syndrome coronavirus 2 ; Severe acute respiratory syndrome related corona virus 2 ; Wuhan coronavirus ; coronavirus disease 2019 virus ; hCoV19 ; nCoV2 ; genetic testing ; gene testing ; gene-based testing ; coronavirus disease ; COVID ; CoV disease ; corona virus disease ; lateral flow assay ; lateral flow test ; isothermal amplification ;
