Non-invasive prenatal diagnosis of single gene disorders with enhanced relative haplotype dosage analysis for diagnostic implementation

Abstract

Non-invasive prenatal diagnosis of single-gene disorders (SGD-NIPD) has been widely accepted, but is mostly limited to the exclusion of either paternal or de novo mutations. Indeed, it is still difficult to infer the inheritance of the maternal allele from cell-free DNA (cfDNA) analysis. Based on the study of maternal haplotype imbalance in cfDNA, relative haplotype dosage (RHDO) was developed to address this challenge. Although RHDO has been shown to be reliable, robust control of statistical error and explicit delineation of critical parameters for assessing the quality of the analysis have not been fully addressed. We present here a universal and adaptable enhanced-RHDO (eRHDO) procedure through an automated bioinformatics pipeline with a didactic visualization of the results, aiming to be applied for any SGD-NIPD in routine care. A training cohort of 43 families carrying CFTR, NF1, DMD, or F8 mutations allowed the characterization and optimal setting of several adjustable data variables, such as minimum sequencing depth, type 1 and type 2 statistical errors, as well as the quality assessment of intermediate steps and final results by block score and concordance score. Validation was successfully performed on a test cohort of 56 pregnancies. 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 conclusive and correctly inferred fetal genotypes in 94.9% of cases, with no false-negative or false-positive results. 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.

Prenatal Diagnosis of Cystic Fibrosis by Celocentesis

“…In the case of monogenic disease, there are two mains limitation for the development of cffDNA-based prenatal tests: the low level of cffDNA present in maternal circulation and the exclusion of the paternal pathogenetic variant [ 26 ].…”

Haplotype‐based noninvasive prenatal diagnosis of methylmalonic acidemia and the discovery of a recurrent pathogenic haplotype associated with c.609G>A

“…Its advantages include high reliability and adaptability to a variety of single-gene disorders, irrespective of the inheritance pattern or the type of molecular abnormality. 12 Despite the reports of NIPD application for autosomal and X-linked recessive disorders like Phenylketonuria (PKU), Duchenne Muscular Dystrophy (DMD), Cystic Fibrosis (CF), thalassemia, etc., its use in MMA at an early pregnancy is scarce. [13][14][15] The requirement for a proband sample in haplotype phasing limits the broader clinical application of RHDO.…”