Non-invasive prenatal diagnosis using fetal DNA in maternal plasma: a preliminary study for identification of paternally-inherited alleles using single nucleotide polymorphisms

Abstract

Objectives: Single nucleotide polymorphism (SNP) with a mutation can be used to identify the presence of the paternally-inherited wild-type or mutant allele as result of the inheritance of either allele in the fetus and allows the prediction of the fetal genotype. This study aims to identify paternal SNPs located at the flanking regions upstream or downstream from the β-globin gene mutations at CD41/42 (HBB:c.127_130delCTTT), IVS1-5 (HBB:c.92+5G>C) and IVS2-654 (HBB:c.316-197C>T) using free-circulating fetal DNA.

Setting: Haematology Lab, Department of Biomedical Science, University of Malaya.

Participants: Eight couples characterised as β-thalassaemia carriers where both partners posed the same β-globin gene mutations at CD41/42, IVS1-5 and IVS2-654, were recruited in this study.

Outcome measures: Genotyping was performed by allele specific-PCR and the locations of SNPs were identified after sequencing alignment.

Results: Genotype analysis revealed that at least one paternal SNP was present for each of the couples. Amplification on free-circulating DNA revealed that the paternal mutant allele of SNP was present in three fcDNA. Thus, the fetuses may be β-thalassaemia carriers or β-thalassaemia major. Paternal wild-type alleles of SNP were present in the remaining five fcDNA samples, thus indicating that the fetal genotypes would not be homozygous mutants.

Conclusions: This preliminary research demonstrates that paternal allele of SNP can be used as a non-invasive prenatal diagnosis approach for at-risk couples to determine the β-thalassaemia status of the fetus.

Keywords: PUBLIC HEALTH.

Figure 1

(A) Location of six paternal SNPs in the β-globin gene. (B) Location and number of paternal SNPs that present the respective mutation for each couple. Five SNPs (rs 35755129, rs 10768684, rs 74234654, rs 200771769 and rs 10742584) are amplified using β 1F and β 1R, while SNP at rs 7946748 is amplified using β 2F and β 2R. Genotype G/A (mutant/wild-type) refers to guanine or adenine on paternal mutant allele or wild-type allele, respectively. A-adenine, T-thymine, C-cytosine, G-guanine; SNP, single nucleotide polymorphism.

Figure 2

Identification of SNP in the paternal alleles. Top panel refers to the alignment of the haplotype sequence of the couple. The following middle panels (1PM, 1PWt, 1MM, 1MWt) refer to the sequence of the wild-type and mutant allele of couple 1. The bottom panel refers to the sequence of enriched fcDNA from maternal plasma. Double peaks were observed at two locations (rs 10742584 and rs 74234654), indicating the presence of paternal mutant allele. PM, paternal mutant allele; PWt, paternal wild-type allele; MM, maternal mutant allele; MWt, maternal wild-type allele; SNP, single nucleotide polymorphism.

Figure 3

The importance of the fetal DNA enrichment steps. The double peaks (adenine and thymine) were observed in the enriched free-circulating fetal DNA sample, while only a single peak (thymine) was observed in the non-enriched free-circulating fetal DNA sample. This result suggests that enriched fetal DNA extracted from maternal plasma increases the detection sensitivity of paternal SNP by reducing the concentration of free-circulating maternal DNA. SNP, single nucleotide polymorphism.