Development of Non-Invasive Prenatal Diagnosis for Single Gene Disorders

Cell-free fetal DNA (cffDNA) is present in the maternal blood from the early first trimester of gestation and makes up 5%-20% of the total circulating cell-free DNA (cfDNA) in maternal plasma. Its presence in maternal plasma has allowed development of noninvasive prenatal diagnosis for single-gene disorders (SGD-NIPD). This can be performed from 9 weeks of amenorrhea and offers an early, safe and accurate definitive diagnosis without the miscarriage risk associated with invasive procedures. One of the major difficulties is distinguishing fetal genotype in the high background of maternal cfDNA, which leads to several technical and analytical challenges. Besides, unlike noninvasive prenatal testing for aneuploidy, NIPD for monogenic diseases represent a smaller market opportunity, and many cases must be provided on a bespoke, patient- or disease-specific basis. As a result, implementation of SGD-NIPD remained sparse, with most testing being delivered in a research setting. The present project aims to take advantage of the unique French collaborative network to make SGD-NIPD possible for theoretically any monogenic disorder and any family.

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 Покажи пълното описание...

Inclusion Criteria: * pregnant woman with 9 weeks of amenorrhea or more * singleton pregnancy * undergoing invasive PND in a context of family history of SGD involving the following genes : HBB, CFTR, FMR1, SMN1, DMPK, DMD, NF1, HTT, F8, F9, GCK, L1CAM, PKHD1, or undergoing prenatal counselling in a context of maternal history of diabetes MODY-GCK * germinal pathogenic paternal and/or maternal mutations previously identified * age 18 years old or over * signing an informed consent Exclusion Criteria: * at risk of SGD involving a de novo pathogenic mutation in a previous child * woman under legal protection