Abstract
Members of the Ashkenazi Jewish community are at an increased risk for inheritance of numerous genetic diseases such that carrier screening is medically recommended. This paper describes the development and evaluation of 30 TaqMan allelic discrimination qPCR assays for 29 mutations on 2 different high-throughput platforms. Four of these mutations are in the GBA gene and are successfully examined using short amplicons due to the qualitative nature of TaqMan allelic discrimination. Two systems were tested for their reliability (call rate) and consistency with previous diagnoses (diagnostic accuracy) indicating a call rate of 99.04% and a diagnostic accuracy of 100% (+/−0.00%) from one platform, and a call rate of 94.66% and a diagnostic accuracy of 93.35% (+/−0.29%) from a second for 9,216 genotypes. Results for mutations tested at the expected carrier frequency indicated a call rate of 97.87% and a diagnostic accuracy of 99.96% (+/−0.05%). This study demonstrated the ability of a high throughput qPCR methodology to accurately and reliably genotype 29 mutations in parallel. The universally applicable nature of this technology provides an opportunity to increase the number of mutations that can be screened simultaneously, and reduce the cost and turnaround time for accommodating newly identified and clinically relevant mutations.
Highlights
The high prevalence of carriers of recessive mutations in the Ashkenazi Jewish population [1] has made genotyping and carrier screening imperative
Unique clusters indicative of different genotypes were formed based on the signal intensity ratio of the two probes being used (VIC and FAM), predicting the genotypes with an accuracy of 100%
Because TaqMan allelic discrimination is a qualitative calculation, the similarities of the four GBA target sequences to the pseudogene did not affect the clustering other than uniformly shifting all of the clusters either towards the VIC or FAM axis based upon which probe was naturally present in the GBAP gene
Summary
The high prevalence of carriers of recessive mutations in the Ashkenazi Jewish population [1] has made genotyping and carrier screening imperative. The American College of Medical Genetics (ACMG) has advised testing for six additional diseases including Bloom Syndrome, Canavan disease, Familial dysautonomia, Fanconi anemia group C, Mucolipidosis IV, and Niemann-Pick disease type A [4,5]. This has led to an increasing demand for quick and accurate diagnoses, and advances in detecting mutations and determining genotypes [6,7]. The platforms that have been developed for high-throughput genotyping purposes, such as a PCR and matrix-assisted, laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry method [10] or arrayed primer extension (APEX) [11], are labor-intensive
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