Non‐invasive fetal genotyping for maternal alleles with droplet digital PCR: A comparative study of analytical approaches

Abstract

Objectives To develop a flexible droplet digital PCR (ddPCR) workflow to perform non‐invasive prenatal diagnosis via relative mutation dosage (RMD) for maternal pathogenic variants with a range of inheritance patterns, and to compare the accuracy of multiple analytical approaches. Methods Cell free DNA (cfDNA) was tested from 124 archived maternal plasma samples: 88 cases for sickle cell disease and 36 for rare Mendelian conditions. Three analytical methods were compared: sequential probability ratio testing (SPRT), Bayesian and z‐score analyses. Results The SPRT, Bayesian and z‐score analyses performed similarly well with correct prediction rates of 96%, 97% and 98%, respectively. However, there were high rates of inconclusive results for each cohort, particularly for z‐score analysis which was 31% overall. Two samples were incorrectly classified by all three analytical methods; a false negative result predicted for a fetus affected with sickle cell disease and a false positive result predicting the presence of an X‐linked IDS variant in an unaffected fetus. Conclusions ddPCR can be applied to RMD for diverse conditions and inheritance patterns, but all methods carry a small risk of erroneous results. Further evaluation is required both to reduce the rate of inconclusive results and explore discordant results in more detail.

Non-invasive prenatal diagnosis (NIPD): current and emerging technologies

“…Proof-of-concept RMD-based NIPD studies have been carried out using dPCR technologies for several monogenic conditions [69,70] , including β-thalassaemia [71,72] , sickle cell disease [73,74] , monogenic diabetes [75] , haemophilia [76,77] , cystic fibrosis [59,78] , methylmalonic acidaemia [79] , inherited deafness [80,81] , as well as a small bespoke cohort with a number of different inheritance patterns [73] . A ddPCR-based RMD method has also been employed for NIPD of families at risk of spinal muscular atrophy, involving molecular counting of the copies of the SMN1 gene in cfDNA of mothers who carry only one functional copy [82] .…”

“…For instance, the sequential probability ratio test (SPRT), which is also the method commonly used with RHDO, has been the analytical method of choice in several studies [69,71,74,76,77,83] . Z-score, also employed in the context of RMD using ddPCR towards NIPT for fetal aneuploidy [84] , has also been selected for analysis [70,72,79,85] , as has a chi-squared test [81] , and a custom Markov chain Monte-Carlo (MCMC) Bayesian approach using JAGS (just another Gibbs sampler) [75] .…”

“…Of concern is the small but significant number of misclassified and inconclusive results which have been reported in several studies that have applied dPCR to detect maternally inherited variants in cfDNA for NIPD of monogenic conditions [70][71][72]74,75,83,85] . The unknown cause of such incorrect results and the wide range of different statistical approaches which have been applied to the various published studies is somewhat problematic.…”

Highlights of the 26th ISPD annual conference, hosted in a vibrant Canadian city along with the Montreal grand prix 2022

“…7 A further paper, presented as a poster in Montreal, describes the potential application of droplet digital PCR for noninvasive prenatal diagnosis of monogenic conditions. 8 Other key highlights of the meeting included carrier screening, now being widely adopted in the USA but less so in other parts of the world. We heard about the challenges associated with ensuring informed patient consent 9 and considerations around what conditions we should be screening for.…”

“…One of the submitted abstracts described the factors influencing uptake of NIPT and noted that it is not only personal values and beliefs influencing choice but also out‐of‐pocket costs of NIPT that hinder equal access to this technology 7 . A further paper, presented as a poster in Montreal, describes the potential application of droplet digital PCR for noninvasive prenatal diagnosis of monogenic conditions 8 . Other key highlights of the meeting included carrier screening, now being widely adopted in the USA but less so in other parts of the world.…”