Rapid Genotyping of ApoL1 Risk Alleles using CRISPR-Cas12a

使用 CRISPR-Cas12a 对 ApoL1 风险等位基因进行快速基因分型

• 批准号: 10384222
• 负责人: Christopher P Larsen
• 金额: $ 25.31万
• 依托单位: NEPHROPATHOLOGY ASSOCIATES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-15 至 2023-03-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10384222
• 关键词: 2019-nCoV; AIDS-Associated Nephropathy; APOL1 gene; Affect; African; African American; African Trypanosomiasis; Alleles; Allografting; Apolipoproteins; Base Sequence; Binding; Binding Proteins; Biological Assay; Blood Circulation; C-terminal; COVID-19; Cells; Chronic; Chronic Kidney Failure; Cicatrix; Clinical; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; DNA sequencing; Detection; Development; Diagnostic; Dialysis procedure; Donor person; Double-Blind Method; End stage renal failure; Equipment; Exhibits; Focal Segmental Glomerulosclerosis; Frequencies; Genes; Genetic Variation; Genomic DNA; Genotype; HIV; Health; High Density Lipoproteins; Human; Incidence; Individual; Infection; Innate Immune System; Kidney; Kidney Diseases; Kidney Glomerulus; Kidney Transplantation; Laboratories; Life; Link; Longevity; Mediating; Membrane Glycoproteins; Methods; Molecular; Parasites; Pathologic; Patients; Phase; Plasma Proteins; Population; Prevalence; Proteins; Public Health; Reagent; Reporting; Resistance; Risk; Sampling; Sensitivity and Specificity; Serum; Severities; Specificity; Techniques; Testing; Therapeutic Agents; Time; Trypanosoma; Trypanosoma brucei brucei; United States; Variant; Virus; Virus Diseases; base; coronavirus disease; cost; diagnostic accuracy; ds-DNA; experience; functional loss; genetic testing; genetic variant; high risk; isothermal amplification; lateral flow assay; loss of function; novel therapeutics; nuclease; point of care; pressure; renal damage; risk variant; skills; tool

SUMMARY. 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 a plasma protein protective against ‘African sleeping sickness’ caused by the parasite Trypanosoma brucei. There are three main allelic variants of APOL1: G0 (wild-type), G1, and G2. The G1 and G2 APOL1 alleles (i.e., renal risk 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 G2 variants of APOL1 are also present at relatively high frequencies among African Americans, with approximately 35% of the African American population having at least one G1 or G2 allele. Despite providing an advantage in survival 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 alleles or are doubly heterozygous for these alleles (G1/G2), have an APOL1 ‘risk genotype’ and are at elevated risk for developing focal segmental glomerulosclerosis (FSGS), which leads to progressive scarring and loss of function of glomeruli. Moreover, the risk genotype is associated with reduced allograft longevity in kidneys transplanted from donors with two risk alleles. More recently, it has been found that other glomerulopathies linked to viral infections, including HIV and SARS-CoV-2, are exacerbated by having the APOL1 risk genotype. Given that approximately 13% of African Americans have a genotype with two risk alleles, APOL1-linked kidney disease represents a potentially massive, yet still underappreciated, public health issue. The available methods for detecting pathological APOL1 variants, including gene sequencing and TaqMan, are relatively expensive and require specialized equipment and skills. Recently, CRISPR/Cas-based methods of detecting specific nucleic acid sequences have been developed. These methods are both simple and inexpensive and therefore offer significant advantages to conventional genotyping methods. In this Phase I application, we propose to develop a 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 to simple and affordable ApoL1 genotyping that is less reliant on specialized equipment. Two novel therapeutic agents for treatment of APOL1-mediated kidney disease are currently in clinical trials, highlighting the urgency to develop better diagnostic tools that can identify individuals who could benefit from these treatments.

摘要：非裔美国人受慢性和终末期肾病 (ESRD) 的影响尤为严重； 虽然 35% 的透析患者是非裔美国人，但美国人口中只有 13.2% 是非裔美国人。 造成这种差异的一个主要因素是载脂蛋白 L1 (APOL1) 的遗传变异。 血浆蛋白可预防布氏锥虫引起的“非洲昏睡病”。 是 APOL1 的三个主要等位基因变体：G0（野生型）、G1 和 G2 G1 和 G2 APOL1 等位基因（即肾型）。 风险等位基因）赋予对昏睡病的抵抗力，而 G0 等位基因则使寄生虫能够存活和感染。 因此，G1 和 G2 等位基因在非洲血统的个体中非常普遍。 APOL1 变体在非裔美国人中也以相对较高的频率出现，大约有 35% 的非裔美国人至少具有一个 G1 或 G2 等位基因，尽管在这方面具有优势。 从非洲锥虫病中存活下来，这些基因型变异使个体容易患上严重的、 具有两个风险等位基因的人，即 G1 或 G2 等位基因纯合的人。 或者这些等位基因 (G1/G2) 是双杂合的，具有 APOL1“风险基因型”并且风险较高 用于发展局灶节段性肾小球硬化症 (FSGS)，这会导致进行性疤痕形成和肾小球功能丧失 此外，风险基因型与肾脏同种异体移植寿命缩短有关。 最近，发现其他肾小球疾病与此相关。 APOL1 风险基因型会加剧病毒感染（包括 HIV 和 SARS-CoV-2）的风险。 大约 13% 的非洲裔美国人的基因型带有两个风险等位基因，即 APOL1 相关肾病 代表了一个潜在的巨大但仍被低估的公共卫生问题。 检测病理性 APOL1 变异，包括基因测序和 TaqMan，相对昂贵且 最近，基于 CRISPR/Cas 的检测特定核酸的方法。 这些方法既简单又便宜，因此已经开发出来。 在此第一阶段的应用中，我们建议开发传统基因分型方法的显着优势。 基于 CRISPR/Cas12a 的概念验证基因分型测定，用于检测 ApoL1 的 G0、G1 和 G2 变体。 一旦开发和优化，该测定将产生一套试剂和技术，以扩大对 简单且经济实惠的 ApoL1 基因分型，较少依赖于专用设备 两种新颖的治疗方法。 用于治疗 APOL1 介导的肾脏疾病的药物目前正在进行临床试验，凸显了紧迫性 开发更好的诊断工具来识别可以从这些治疗中受益的个体。