Fast Temperature-Gradient COLD PCR for the enrichment of the paternally inherited SNPs in cell free fetal DNA; an application to non-invasive prenatal diagnosis of β-thalassaemia.

OBJECTIVE:To develop a sensitive, specific, simple, cost-effective and reproducible platform for the non-invasive prenatal detection of paternally inherited alleles for β-thalassaemia. The development of such an assay is of major significance in order to replace currently-applied invasive methods containing inherent fetal loss risks. METHODS:We present a fast Temperature-Gradient Co-amplification at Lower Denaturation Temperature Polymerase Chain Reaction (fast TG COLD PCR) methodology for the detection of the paternally-inherited fetal alleles in maternal plasma. Two single-nucleotide polymorphisms (SNPs), rs7480526 (G/T) and rs968857 (G/A) that are located on the β-globin gene cluster and exhibit a high degree of heterozygosity in the Cypriot population were selected for evaluation. Seventeen maternal plasma samples from pregnancies at risk for β-thalassemia were analysed for the selected SNPs using the novel fast TG COLD PCR assay. RESULTS:Using fast TG COLD PCR, the paternally inherited allele in cell free fetal DNA was correctly determined for all the 17 maternal plasma samples tested, showing full agreement with the Chorionic Villus Sampling (CVS) analysis. CONCLUSIONS:Our findings are encouraging and demonstrate the efficiency and sensitivity of fast TG COLD PCR in detecting the minor paternally-inherited fetal alleles in maternal plasma for the development of a NIPD assay for β-thalassaemia

Fast Temperature-Gradient COLD PCR for rs7480526.

<p>The input conventional PCR products resulted from spiked genomic DNA amplification consisting of 95% maternal alleles (blue) and 5% fetal alleles (red). First, the PCR products are subjected to a number of cycles of regular PCR to produce an initial pool of target amplicons. Next, the denaturation temperature is set to the first Tc, which is lower than the Tm of the maternal allele. Since the melting temperature of the fetal alleles is lower than that of the maternal alleles, fetal alleles get substantially denatured whereas the maternal alleles remain substantially double-stranded. Then we decrease the temperature for primer annealing and extension. Next, we move on to the second critical temperature (Tc2) and repeat the same procedure for ten cycles. In that way we span a range of three (SNP rs968857) or four (SNP rs7480526) critical temperatures and so we assure the preferential enrichment of the minor fetal allele and increase its abundance within the final product.</p

Minor allelic frequency of the 17 maternal plasma samples analyzed for all the 141 replicates.

<p>Blue columns: replicate samples with expected minor allele (Mother: GG/ Fetus: GA or GT). A minor allelic frequency distribution between 21–100% was observed in 101 out of the 105 replicates. A minor allelic frequency between 0–10% was observed in 2 out of 105 replicates and 11–20% in 2 out of 105 replicates. Red columns: replicate samples with no expected minor allele (Mother: GG/ Fetus: GG). A minor allelic frequency below 10% was observed in 33 out of the 36 replicates. A minor allelic frequency between 11–20% was observed in 2 out of 36 replicates and 61–70% minor allelic frequency in 1 out of 36 replicates.</p

Electropherograms of spiked genomic DNA for SNPs rs7480526 and rs968857 using fast-COLD-PCR and fast-TG-COLD-PCR.

<p>The top sequence above each electropherogram presents the reference sequence based on the GRCh37 genome assembly. Enrichment of the minor allele is observed using the fast TG COLD PCR whereas non enrichment is observed with fast COLD PCR for the same samples.</p

Primer sequences and fragment sizes for pre-amplification and fast Temperature-Gradient COLD PCR.

<p>Primer sequences and fragment sizes for pre-amplification and fast Temperature-Gradient COLD PCR.</p

Electropherograms of maternal plasma of families 182, 197 for rs7480526, rs968857 respectively, after fast-TG-COLD-PCR/conventional-PCR.

<p>Three runs are indicated, 3 replicates per run, 9 reactions total. The top sequence above each electropherogram presents the reference sequence based on the GRCh37 genome assembly. Enrichment of the paternally inherited minor allele is observed in all replicates for SNP rs7480526 and in 8 out of 9 reactions for SNP rs968857 with fast TG COLD PCR as opposed to no enrichment with conventional PCR.</p

Implementation of High Resolution Whole Genome Array CGH in the Prenatal Clinical Setting: Advantages, Challenges, and Review of the Literature

Array Comparative Genomic Hybridization analysis is replacing postnatal chromosomal analysis in cases of intellectual disabilities, and it has been postulated that it might also become the first-tier test in prenatal diagnosis. In this study, array CGH was applied in 64 prenatal samples with whole genome oligonucleotide arrays (BlueGnome, Ltd.) on DNA extracted from chorionic villi, amniotic fluid, foetal blood, and skin samples. Results were confirmed with Fluorescence In Situ Hybridization or Real-Time PCR. Fifty-three cases had normal karyotype and abnormal ultrasound findings, and seven samples had balanced rearrangements, five of which also had ultrasound findings. The value of array CGH in the characterization of previously known aberrations in five samples is also presented. Seventeen out of 64 samples carried copy number alterations giving a detection rate of 26.5%. Ten of these represent benign or variables of unknown significance, giving a diagnostic capacity of the method to be 10.9%. If karyotype is performed the additional diagnostic capacity of the method is 5.1% (3/59). This study indicates the ability of array CGH to identify chromosomal abnormalities which cannot be detected during routine prenatal cytogenetic analysis, therefore increasing the overall detection rate. In addition a thorough review of the literature is presented