Next generation sequencing of SNPs for non-invasive prenatal diagnosis: challenges and feasibility as illustrated by an application to β-thalassaemia

Abstract

b-Thalassaemia is one of the most common autosomal recessive single-gene disorder worldwide, with a carrier frequency of 12% in Cyprus. Prenatal tests for at risk pregnancies use invasive methods and development of a non-invasive prenatal diagnostic (NIPD) method is of paramount importance to prevent unnecessary risks inherent to invasive methods. Here, we describe such a method by assessing a modified version of next generation sequencing (NGS) using the Illumina platform, called 'targeted sequencing', based on the detection of paternally inherited fetal alleles in maternal plasma. We selected four single-nucleotide polymorphisms (SNPs) located in the b-globin locus with a high degree of heterozygosity in the Cypriot population. Spiked genomic samples were used to determine the specificity of the platform. We could detect the minor alleles in the expected ratio, showing the specificity of the platform. We then developed a multiplexed format for the selected SNPs and analysed ten maternal plasma samples from pregnancies at risk. The presence or absence of the paternal mutant allele was correctly determined in 27 out of 34 samples analysed. With haplotype analysis, NIPD was possible on eight out of ten families. This is the first study carried out for the NIPD of b-thalassaemia using targeted NGS and haplotype analysis. Preliminary results show that NGS is effective in detecting paternally inherited alleles in the maternal plasma.

Thalassemia 2016: Modern medicine battles an ancient disease

“…The methodology was also applied to deletional a-thalassemia but not sufficiently accurate for most bglobin point mutations. Recently, more modern techniques of DNA analysis using next generation sequencing (NGS) were applied toward the goal of noninvasive fetal diagnosis of thalassemia [14]. Using this technique, maternal peripheral blood is drawn at 9 to 10 weeks, before chorionic villus sampling can be performed, and analysis is performed for a panel of single nucleotide polymorphisms (SNPs) which are predetermined, using family studies, to be linked to the paternal mutant allele.…”

“…Using the technique of NGS, the use of four SNP pairs has been found to be sufficiently accurate to determine if the fetus is affected. Thus far the technique is only in experimental use since it is not diagnostic in all cases and it has required confirmation by CVS testing [14]. Yet, it is probable that in the near future, this innovative technique will be perfected such that it will be in routine use.…”

Non‐invasive prenatal diagnosis of Duchenne and Becker muscular dystrophies by relative haplotype dosage

“…As part of the non‐invasive prenatal diagnosis for single gene disorders (NIPSIGEN) project conducted at Birmingham Women's National Health Service (NHS) Foundation Trust (UK), we aimed at developing an affordable NIPD test for SGDs. After carefully considering various methods described in previous studies, we decided to adopt the relative haplotype dosage (RHDO) analysis developed by Lo and colleagues . In 2010, Lo was able to construct a genome‐wide genetic map of the fetus from maternal plasma DNA sequences using RHDO and subsequently demonstrated how this could be applied for NIPD of β‐thalassemia and CAH .…”

Birth of children with severe β‐thalassemia at a tertiary obstetric hospital: what are the reasons behind it?

“…The possibility to retrieve fetal DNA from maternal plasma has made available a new source of fetal genetic material for noninvasive analysis of numerous fetal pathological conditions, thus avoiding or reducing the use of invasive techniques for prenatal diagnosis . β‐thalassemia has been chosen as a model of clinically relevant genetic disease …”