Fetal Genotyping in Maternal Blood by Digital PCR: Towards NIPD of Monogenic Disorders Independently of Parental Origin

<div><p>Purpose</p><p>To date, non-invasive prenatal diagnosis (NIPD) of monogenic disorders has been limited to cases with a paternal origin. This work shows a validation study of the Droplet Digital PCR (ddPCR) technology for analysis of both paternally and maternally inherited fetal alleles. For the purpose, single nucleotide polymorphisms (SNPs) were studied with the only intention to mimic monogenic disorders.</p><p>Methods</p><p>NIPD SNP genotyping was performed by ddPCR in 55 maternal plasma samples. In 19 out of 55 cases, inheritance of the paternal allele was determined by presence/absence criteria. In the remaining 36, determination of the maternally inherited fetal allele was performed by relative mutation dosage (RMD) analysis.</p><p>Results</p><p>ddPCR exhibited 100% accuracy for detection of paternal alleles. For diagnosis of fetal alleles with maternal origin by RMD analysis, the technology showed an accuracy of 96%. Twenty-nine out of 36 were correctly diagnosed. There was one FP and six maternal plasma samples that could not be diagnosed.</p><p>Discussion</p><p>In this study, ddPCR has shown to be capable to detect both paternal and maternal fetal alleles in maternal plasma. This represents a step forward towards the introduction of NIPD for all pregnancies independently of the parental origin of the disease.</p></div

Results of Z-Score values.

<p>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.</p

Clinical interpretation algorithm where Allele 1 (1) represents the maternal healthy allele and Allele 2 (2) represents the maternal mutated allele.

<p>Clinical interpretation algorithm where Allele 1 (1) represents the maternal healthy allele and Allele 2 (2) represents the maternal mutated allele.</p

Primer and probe sequences and ddPCR conditions.

<p>This figure also includes optimal Temperature of Annealing (Ta) for each Taqman assay for the multiplex <i>SRY/GAPDH</i> and <i>RASSSF1A/GAPDH</i> assays.</p

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.

<p>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).</p

Identification of chromosomal rearrangements in WAGR locus in syndromic and non-syndromic patients with aniridia.

<p>Targeted array-based comparative genomic hybridization (aCGH) analysis identified deletions of different sizes ranging from3.3 Kb to 13.4 Mb. The red bars show intragenic <i>PAX6</i> deletions in two patients with isolated aniridia, ANIRIDIA-039 (chr11:31,820,789–31,824,052) and ANIRIDIA-052 (chr11:31,760,458–31,823,847). The green bars show 3’ upstream deletions affecting 3' regulatory regions of <i>PAX6</i>, identified in three families with isolated aniridia: ANIRIDIA-008 (chr11:31,147,306–31,714,853), ANIRIDIA-021 (chr11:31,186,493–31,698,208) and ANIRIDIA-067 (chr11:31,083,877–31,704,548). Purple bars show large deletions affecting several contiguous genes, in two patients with WAGR (ANIRIDIA-020, chr11:29,750,813–32,752,091), and WAGRO (ANIRIDIA-070, chr11:21,586,131–33,168,232) syndromes. Genes delineating both syndromes are highlighted in red. The blue bar represents a novel gene contiguous deletion syndrome involving <i>PAX6</i> and 45 upstream genes in a syndromic patient with aniridia (ANIRIDIA-064, chr11:18,536,224–31,923,308). Genomic coordinates are shown in the x-axis and are based on the Human Genome Assembly hg19.</p

Identification of intragenic <i>PAX6</i> deletion in patients with isolated aniridia.

<p>Targeted array-based comparative genomic hybridization (aCGH) analysis identified two deletions involving partial <i>PAX6</i> deletions in two patients. Colored bars represent the genomic positions of the deletions. Schematic representation of the complete intron-exon structure of <i>PAX6</i> is shown. Exons are indicated by colored rectangles that are wider for the coding regions. CGH array data for both individuals is shown. The patient <i>versus</i> reference log2-ratio for the relative hybridization intensities of probes is plotted. Dots with log2-ratio around -1 indicate a heterozygous deletion (green dots), log2-ratio 0 indicates a normal pattern, and +0.6 indicates a heterozygous amplification (red dots). Shaded areas indicate deletions. Genomic coordinates are shown in the x-axis and are based on the Human Genome Assembly hg19. The red bar indicates a ~63 kb deletion encompassing from exon 5a to exon 13 of <i>PAX6</i> found in patient ANIRIDIA-052 (chr11:31,760,458–31,823,847). The grey bar represents a ~3.3 kb deletion encompassing from exon 5a to exon 7 of <i>PAX6</i> gene found in patient ANIRIDIA-039 (chr11:31,820,789–31,824,052).</p

Patients with isolated or syndromic aniridia carrying chromosomal deletions in 11p13.

<p>Patients with isolated or syndromic aniridia carrying chromosomal deletions in 11p13.</p

Identification of 3’ regulatory deletions of <i>PAX6</i> in patients with isolated aniridia.

<p>Targeted array-based comparative genomic hybridization (aCGH) analysis identified deletions involving telomeric deletions to <i>PAX6</i> in two patients ANIRIDIA-008 and ANIRIDIA-021. Patient ANIRIDIA-067 was used as positive control for validation purposes. The colored bars represent the genomic positions of the deletions. The red asterisks indicate a cluster of <i>PAX6</i> regulatory regions located in intronic positions of <i>ELP4</i>, as reviewed by Bathia, <i>et al</i>, 2013. Exons are indicated by colored rectangles that are wider for the coding regions. CGH array data for the two patients with previously unknown 3' telomeric <i>PAX6</i> deletions are shown. The patient <i>versus</i> reference log2-ratio for the relative hybridization intensities of probes is plotted. Dots with log2-ratio around -1 indicate a heterozygous deletion (green dots), log2-ratio 0 indicates a normal pattern and +0.6 indicates a heterozygous amplification (red dots). Shaded areas indicate deletions. Genomic coordinates are shown in the x-axis and are based on the Human Genome Assembly hg19. The grey bar indicates a ~567 kb deletion in patient ANIRIDIA-008 (chr11:31,147,306–31,714,853). The orange bar indicates a ~511 kb deletion in patient ANIRIDIA-021 (chr11:31,186,493–31,698,208).</p