Evaluation of the Diagnostic Performance of Non-Invasive Prenatal Diagnosis for Single Gene Disorders

Since the identification of cffDNA in maternal plasma in 1997, there have been rapid developments in exploiting its presence for prenatal diagnosis and screening. The first proof of principle studies using cfDNA from maternal plasma to detect fetal aneuploidy were published in 2008 following which there was rapid commercialization. Nowadays, non-invasive prenatal testing (NIPT) for aneuploidies is widely used across the world as a screening test for the most frequent fetal trisomies. Unlike non-invasive prenatal testing, where a positive result requires confirmation following an invasive test, non-invasive prenatal diagnosis (NIPD) offers the advantage of a definitive diagnosis without an invasive procedure - and its associated miscarriage risk - because confined placental mosaicism does not occur with NIPD for single-gene disorders (SGD).

NIPD can be offered earlier in pregnancy than invasive testing, from 7 weeks of gestation. This can reduce parental anxiety and allows more time for decision-making and planning. Indeed, there are substantial challenges to overcome for SGD-NIPD. i/ Circulating cffDNA, which is released from the placenta from about 4 weeks gestation, makes up only 5%-20% of total circulating cfDNA in maternal plasma. This percentage increases with gestation and is influenced by factors such as maternal weight, smoking, and pregnancy complications such as preeclampsia. Consequently, optimized techniques and highly sensitive detection approaches are required to detect variants in the fetal DNA. ii/ fetal fraction must be calculated to confirm that there are sufficient levels of cffDNA present and to avoid false negative results. iii/ Another issue is the short fragment length of cffDNA, which makes detection of triplet repeats and large deletions or duplications challenging.

Besides fetal sex determination and fetal Rhesus D status, principal investigator's team was the first to propose SGD-NIPD for use in clinical practice in France for autosomal disorders caused by de novo or paternally inherited mutations, for which variants in the fetal DNA can easily be distinguished in the high background of maternal cfDNA. Droplet-digital Polymerase Chain Reaction or Next-Generation Sequencing can be used to target a single mutation for this analysis.

However, this approach requires mutation-specific developments and is restricted to point mutations that are absent from maternal DNA. NIPD for X-linked disorders, as well as autosomal dominant maternally inherited or autosomal recessive disorders for which both parents are carriers of the same mutation has posed a greater challenge. A quantitative approach is needed to ascertain fetal inheritance of the maternal mutation. In autosomal dominant diseases with maternal mutation or autosomal recessive diseases, the ratio between the total copies of the mutant allele (M) and the wildtype allele (N) in the maternal plasma contributed by both the maternal and fetal cell-free DNA is expected to be balanced (M=N) if the genotype of the fetus (M/N) is identical to the mother (M/N). However, the allelic ratio will be imbalanced if the fetal genotype is different from the maternal genotype. If the fetus has inherited both parental wildtype alleles (N/N), there would be additional dosage of the wildtype allele in the maternal plasma contributed from the fetus, resulting in under-representation in the total copies of the mutant allele (M<N). Conversely, if the fetus has inherited the parental mutant alleles (M/M), there would be additional dosage of the mutant allele in the maternal plasma contributed from the fetus, resulting in over-representation in the total copies of the mutant allele (M>N). The degree of expected allelic imbalance in maternal plasma depends on the DNA fetal fraction in the maternal plasma.

A quantitative relative mutation dosage (RMD) approach has been developed to detect such mutant allelic imbalance. This approach has been applied to the non-invasive detection of recessive disorders such as beta-thalassemia and sickle cell anemia but also for X-linked disorders like hemophilia . Nevertheless, direct interrogation of the mutation appears to be difficult - even impossible - in certain genomic loci due to the presence of repetitive sequences, homologous pseudogenes, and undefined genomic rearrangement. Moreover, successful classification of allelic imbalance is statistically dependent on the available copies of mutant and wildtype alleles in the blood sample, hampered by the very low absolute concentration of cfDNA. As a result, RMD analysis is still at a proof-of-concept phase, being evaluated in a limited number of studies, with a limited number of patients, and has never been implemented in standard care diagnosis to investigator's knowledge.