Non-invasive prenatal diagnosis (NIPD): how analysis of cell-free DNA in maternal plasma has changed prenatal diagnosis for monogenic disorders

Abstract

Cell-free fetal DNA (cffDNA) is released into the maternal circulation from trophoblastic cells during pregnancy, is detectable from 4 weeks and is representative of the entire fetal genome. The presence of this cffDNA in the maternal bloodstream has enabled clinical implementation of non-invasive prenatal diagnosis (NIPD) for monogenic disorders. Detection of paternally inherited and de novo mutations is relatively straightforward, and several methods have been developed for clinical use, including quantitative polymerase chain reaction (qPCR), and PCR followed by restriction enzyme digest (PCR-RED) or next-generation sequencing (NGS). A greater challenge has been in the detection of maternally inherited variants owing to the high background of maternal cell-free DNA (cfDNA). Molecular counting techniques have been developed to measure subtle changes in allele frequency. For instance, relative haplotype dosage analysis (RHDO), which uses single nucleotide polymorphisms (SNPs) for phasing of high- and low-risk alleles, is clinically available for several monogenic disorders. A major drawback is that RHDO requires samples from both parents and an affected or unaffected proband, therefore alternative methods, such as proband-free RHDO and relative mutation dosage (RMD), are being investigated. cffDNA was thought to exist only as short fragments (<500 bp); however, long-read sequencing technologies have recently revealed a range of sizes up to ∼23 kb. cffDNA also carries a specific placental epigenetic mark, and so fragmentomics and epigenetics are of interest for targeted enrichment of cffDNA. Cell-based NIPD approaches are also currently under investigation as a means to obtain a pure source of intact fetal genomic DNA.

Noninvasive Prenatal Screening for Single-Gene Disorders

“…Maternal genomic DNA is accounted for during the analysis in cfDNA for SGDs, and confined placental mosaicism is extremely rare in monogenic conditions. 13 In the case of positive cfDNA results for SGD in low-risk pregnancies, confirmation with a diagnostic test should be recommended before irreversible decisions in pregnancy management are made. Irrespective of the initial indication for testing, features of the specific monogenic condition should be discussed, including penetrance, expressivity, clinical course, and prognosis.…”

“…Several polymerase chain reaction (PCR)-based methods have been used, including real-time quantitative PCR for fetal sex determination. 13 Digital PCR has also recently been used to identify male fetuses in families at risk for X-linked disorders 14 and for paternal variant exclusion. Initially, prenatal cfDNA screening of SGDs utilized PCR, followed by restriction enzyme digestion, specifically for the diagnosis of FGFR3-related achondroplasia 15 and thanatophoric dysplasia.…”

“…16 Targeted sequencing through PCR, followed by next-generation sequencing (PCR-NGS), has been shown to be an improved method when compared with traditional methods, allowing the detection of multiple variants. 13 PCR-NGS has been used for the detection of fetal ABO blood group 17 and some autosomal recessive disorders. 18 For the latter, PCR-NGS is used to detect or exclude the paternal variant.…”

“…Early intervention and therapies are available for some of these disorders, with the potential to improve both short-term and long-term outcomes and quality of life for affected individuals. 13 There are several potential disadvantages of cfDNA screening for SGDs beyond the lack of guidelines and clinical validity. In the setting of a positive result in a low-risk pregnancy, it may be challenging to find a laboratory that is willing or able to test for the specific variant on chorionic villi and/or amniotic fluid specimens.…”

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

“…By relying on indirect diagnosis, RHDO theoretically allows diagnosis for any family, regardless of the inheritance pattern or the type of molecular abnormality and has been successfully applied to several SGDs [ 17 – 21 ]. However, to our knowledge, clinical implementation in public health service laboratories around the world remains sparse, probably because quality controls and decision thresholds remain complex to define [ 22 – 24 ].…”