Abstract
Clinical genetic testing often takes days to weeks, but rapid, affordable tests during outpatient visits could significantly benefit patients. This is crucial for detecting common, actionable point mutations, such as those linked to hereditary transthyretin (TTR) amyloidosis, which is often underdiagnosed in individuals of West African ancestry with congestive heart failure. Here we developed a method for detecting known DNA variants using allele-specific polymerase chain reaction (ASPCR) and electrical impedance. Oligonucleotide primers were designed to selectively amplify the ancestral and variant allele of TTR c.424G>A (p.Val142Ile). PCR products were detected in solution using passive-flow microfluidic impedance cytometry across multiple excitation frequencies. Impedance responses correlated with DNA concentration, allowing for the calculation of a robust DNA quantification score. TTR V142I genotypes from six patients (four heterozygous, two wildtype) matched results from gel electrophoresis and sequencing. Future improvements will focus on reducing DNA input requirements and enabling multiplex variant detection.
Introduction
Most clinical genetic tests are characterized by slow turnaround times (TAT), often spanning from days to weeks. Both clinicians and patients could benefit from genetic testing that is rapid, affordable, and easy-to-interpret. This is particularly important for relatively common and clinically significant DNA variants, such as those linked to hereditary transthyretin amyloidosis (ATTR), cystic fibrosis or sickle cell anemia. ATTR is a treatable cause of heart failure with a hereditary form that disproportionally affects patients of West African ancestry1,2. The TTR variant c.424G>A, p.V142I (aka V122I) is pathogenic and occurs in 3–4% of individuals of West African ancestry. This condition profoundly affects individuals and their communities, leading to diminished quality of life, higher rates of morbidity and mortality, and a substantial strain on healthcare systems. Despite a significant association with heart failure, clinical manifestations often precede diagnosis by years3,4. V142I ATTR remains underdiagnosed in the US and other countries due to the lack of available information about the variant’s clinical significance, coupled with the high cost of genetic diagnosis. Timely ATTR diagnosis is important now that effective ATTR-specific therapies are approved5,6 or in clinical trials7,8,9,10,11. In response to this unmet need, we previously developed a rapid molecular test to obtain a genetic diagnosis for TTR V142I from a drop of blood in less than 30 min12,13. Our approach combines allele-specific polymerase chain reaction (ASPCR)14,15 and rapid gel electrophoresis to identify patients that are positive V142I heterozygous or homozygous, or negative (wildtype). The rapid TTR assay was employed in post-hoc analyses of a congestive heart failure (CHF) clinical trial, which found that 11% of African-Americans carried V142I in the TOPCAT trial, suggesting under-recognition of TTR-related heart failure in this cohort16.