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.

摘要非裔美国人不成比例地受到慢性和终末期肾病（ESRD）的影响; 虽然35%的透析患者是非洲裔美国人，但美国人口中只有13.2%是非洲裔美国人。 导致这种差异的一个主要因素是载脂蛋白L1（APOL 1）的遗传变异。APOL 1是一种 血浆蛋白，可预防由寄生虫布氏锥虫引起的“非洲昏睡病”。那里 APOL 1有三种主要的等位基因变体：G 0（野生型）、G1和G2。G1和G2 APOL 1等位基因（即，肾 风险等位基因）赋予抵抗昏睡病，而G 0等位基因使寄生虫存活和感染。 因此，G1和G2等位基因在非洲血统的个体中非常普遍。G1和G2 APOL 1的变体在非裔美国人中也以相对高的频率存在， 35%的美国黑人至少有一个G1或G2等位基因。尽管提供了优势， 从非洲锥虫存活，这些基因型变异使个体易患严重的， 无法治愈的肾病具有两个风险等位基因的人，即，G1或G2等位基因纯合的人 或这些等位基因（G1/G2）双杂合，具有APOL 1“风险基因型”， 发展局灶性节段性肾小球硬化症（FSGS），导致进行性瘢痕形成和 肾小球的功能此外，风险基因型与肾脏移植物寿命缩短有关 从有两个危险等位基因的捐赠者身上移植的最近，人们发现其他肾小球疾病与 包括HIV和SARS-CoV-2在内的病毒感染，因具有APOL 1风险基因型而加剧。给定 大约13%的非裔美国人有一个基因型，有两个危险等位基因，APOL 1连锁肾病 代表了一个潜在的巨大的，但仍然被低估的，公共卫生问题。可用的方法 检测病理性APOL 1变体，包括基因测序和TaqMan，相对昂贵， 需要专门的设备和技能。最近，基于CRISPR/Cas的检测特异性核酸的方法， 已经开发了酸性序列。这些方法既简单又便宜，因此提供 与传统的基因分型方法相比具有显著的优势。在第一阶段的申请中，我们建议开发 一种基于CRISPR/Cas 12 a的基因分型方法，用于检测ApoL 1的G 0、G1和G2变体。 一旦开发和优化，这种检测将导致一套试剂和技术，以扩大获得 简单且经济实惠的ApoL 1基因分型，对专业设备的依赖性较低。两种新的治疗方法 用于治疗APOL 1介导的肾病的药物目前正在临床试验中，这突出了治疗APOL 1介导的肾病的紧迫性。 开发更好的诊断工具，以识别可能从这些治疗中受益的个体。