Figure 1.
Strategy for the non-invasive prenatal exclusion of homozygous (−−SEA) deletion in maternal plasma.
Schematic drawing showing the basic principle of our strategy for the non-invasive prenatal exclusion of homozygous (−−SEA) deletion based on SNP typing linked to the paternal-normal α-globin allele. The normal α-globin gene cluster and corresponding deleted region of (−−SEA) allele are denoted by the colored box with black line and the orange dotted line, respectively. The at-risk heterozygous couples sharing the same (−−SEA) deletion are shown on the top and the possible consequences of NIPD for the fetus are demonstrated by the bottom panel of this figure. The arrows indicate an informative SNP identified within the deletion breakpoints that can be used to differentiate between the mother's (with T allele, downward arrows) and the father's (with C allele, upward arrows) normal allele. If C allele is not detected in the cffDNA fraction of maternal plasma, the fetus may either be heterozygous, those who inherit a normal maternal T allele and a deleted paternal allele, or homozygous for the (−−SEA) allele, those who inherit deleted alleles from both parents. In contrast, the presence of C allele in cffDNA denotes that the fetus inherits the normal paternal allele, and therefore, we can exclude the possibility of Hb Bart's syndrome for the fetus.
Figure 2.
NIPD flow chart for exclusion of a large fetal deletion from cfDNA in maternal plasma.
Table 1.
The oligonucleotide sequences of Multiplex PCR amplification and extension primer sequences for the mini-sequencing reaction.
Table 2.
Allele frequencies of 16 SNP markers in 150 normal unrelated individuals and 150 heterozygote samples.
Figure 3.
The sensitivity and specificity of allele-specific real-time PCR method used for NIPD.
Panel A shows one set of representative data from analysis of g.31921 T>C marker by allele-specific real-time PCR, in which the SNP profiling of artificial model samples is exported by amplification blot (left) and dissociation curve analysis (right). Our results showed superior sensitivity and specificity in all dilutions (see materials and methods), indicated by the arrows for each test, with a sensitivity of detection of 2 copies of the target sequence. Panel B shows the quantitative difference between the respective CT values (ΔCT) of maternal alleles (CT, maternal) and paternal alleles (CT, paternal) in analysis of g.31921T>C artificial model samples (see Materials and Methods). These results indicated a clear discrimination of the paternal allele from the maternal allele on experimental serial dilutions. Following the ΔCT,(paternal-maternal) analysis, the maternal allele cluster is above a ΔCT value of 6, while the paternal allele cluster is at a value of less than 6, thus the arbitrarily assigned ΔCT cut-off value (gray dotted line) was used to distinguish between the presence of maternal from paternal alleles in the present study. Panel C shows the NIPD results obtained from detection of 65 at-risk fetus using 9 SNP markers in cfDNA as paternal-normal marker by allele-specific real-time PCR. Among 65 samples tested, correct appraisal of the presence (n = 33) or absence (n = 30) of the paternal-normal allele. Two cases labeled uncertain due to sample hemolysis by mishandling in the process of blood transport.
Table 3.
Summary of the results of the informative SNP alleles by the analysis of cell free fetal DNA in the maternal plasma.