Non-Invasive Prenatal Diagnosis of Single Gene Disorders with enhanced Relative Haplotype Dosage Analysis for diagnostic implementation

Non-invasive prenatal diagnosis for single-gene disorders (SGD-NIPD) has been widely adopted by patients, but is mostly limited to the exclusion of paternal or de novo mutations. Indeed, it is still difficult to infer the inheritance of maternal allele from cell free DNA (cfDNA) analysis. Based on the study of maternal haplotypes imbalance in cfDNA, relative haplotype dosage (RHDO) was developed to address this challenge. Although RHDO has proven to be reliable, robust control of statistical error and explicit delineation of critical parameters for assessing the quality of analysis have not been fully considered yet. Here we propose a universal and adaptable enhanced-RHDO procedure (eRHDO) through an automated bioinformatics pipeline with a didactical visualization of results that aims to be applied for any SGD-NIPD in routine care. A training cohort of 43 families carriers for CFTR, NF1, DMD, or F8 mutations allowed the characterization and optimal setting of several adjustable data variables, such as minimal sequencing depth and type 1 and type 2 statistical errors, as well as the quality assessment for intermediate steps and final result through block score and concordance score. Validation was successfully carried out on 56 pregnancies of the test cohort. Finally, computer simulations were used to estimate the effect of fetal-fraction, sequencing depth and number of informative SNPs on the quality of results. Our workflow proved to be robust, as we obtained 94.9% conclusive and correctly inferred fetal genotypes, without any false negative or false positive result. By standardizing data generation and analysis, we fully describe a turnkey protocol for laboratories wishing to offer eRHDO-based non-invasive prenatal diagnosis for single-gene disorders as an alternative to conventional prenatal diagnosis.