Fig 1.
Schematic representation of the study design based on a SNP study showing: parental genotypes, the data analysis approach in maternal plasma and the inheritance pattern mimicked.
Parental genotyping combination for allele 1/allele 2 of a certain SNP can be used to represent an inheritance pattern for a point mutation. The different parental genotype combinations and their correspondence with a specific inheritance pattern are detailed in the figure. Analysis of exclusive paternal sequences requires a detection approach while the study of maternal genomic regions requires a Relative Mutation Dosage (RMD) analysis. A simulation of an NIPD for a dominant disease with paternal origin can be done using a case in which the mother is homozygous for an SNP and the father is heterozygous (blue background). Presence/absence of the exclusive paternal allele could be associated with a fetal genotype. On the contrary, a heterozygous mother and a homozygous father will mimic a dominant disorder with a maternal origin (red background). This simulation can be also considered for recessive diseases in which both parents are carriers of a different mutation. NIPD for a recessive disease in which both parents are carriers of the same mutation can be simulated by taking as an example a couple in which the mother and the father are heterozygous for an SNP (yellow background). Finally, the simulation of NIPD for X-linked disorders can be done by a case in which mother is heterozygous for a SNP and the father is hemizygous (green background).
Fig 2.
Primer and probe sequences and ddPCR conditions.
This figure also includes optimal Temperature of Annealing (Ta) for each Taqman assay for the multiplex SRY/GAPDH and RASSSF1A/GAPDH assays.
Fig 3.
This figure shows the results of Z-Score values calculated in all thirty-six cases considered for Relative Mutation Dosage (RMD) to establish the fetal genotyping in maternal plasma samples. Balance and Imbalance allelic ratio was used to ascertain the fetal genotype. Blue circle = Homozygous fetus for Allele 2 (2>1); Blue triangle = Heterozygous Allele 1/ Allele 2 fetus (2 = 1); Blue square = Homozygous fetus for Allele 1 (1>2)and Red square = False Positive.
Fig 4.
Clinical interpretation algorithm where Allele 1 (1) represents the maternal healthy allele and Allele 2 (2) represents the maternal mutated allele.