Development and validation of a novel panel of 16 STR markers for simultaneous diagnosis of \u03b2-thalassemia, aneuploidy screening, maternal cell contamination detection and fetal sample authenticity in PND and PGD/PGS cases

Zohreh Sharifi,Fatemeh Golnabi,Sirous Zeinali,Ameneh Sarhadi Bandehi,Hossein Farahzadi,Atefeh Joudaki,Maryam Abiri,Rasaneh Yazdani,Sanaz Naderi,Yeganeh Keshvar,Shirin Ghadami,Faezeh Rahiminejad,Mehdi Shafaat

doi:10.1038/s41598-019-43892-2

Abstract

Prenatal diagnosis (PND) may be complicated with sample mix-up; maternal cell contamination, non-paternity and allele drop out at different stages of diagnosis. Aneuploidy screening if combined with PND for a given single gene disorder, can help to detect any common aneuploidy as well as aiding sample authenticity and other probable complications which may arise during such procedures. This study was carried out to evaluate the effectiveness of a novel panel of STR markers combined as a multiplex PCR kit (HapScreen™ kit) for the detection of β-thalassemia, aneuploidy screening, ruling in/out maternal cell contamination (MCC), and sample authenticity. The kit uses 7 STR markers linked to β-globin gene (HBB) as well as using 9 markers for quantitative analysis of chromosomes 21, 18, 13, X and Y. Selection of the markers was to do linkage analysis with β-globin gene, segregation analysis and to perform a preliminary aneuploidy screening of fetal samples respectively. These markers (linked to the β-globin gene) were tested on more than 2185 samples and showed high heterozygosity values (68.4–91.4%). From 2185 fetal cases we found 3 cases of non-paternity, 5 cases of MCC, one case of sample mix-up and one case of trisomy 21 which otherwise may have end up to misdiagnosis. This kit was also successfully used on 231 blastomeres for 29 cases of pre-implantation genetic diagnosis (PGD) and screening (PGS). The markers used for simultaneous analysis of haplotype segregation and aneuploidy screening proved to be very valuable to confirm results obtained from direct mutation detection methods (i.e. ARMS, MLPA and sequencing) and aneuploidy screening.

Highlights

Www.nature.com/scientificreports β-thalassemia (β-thal) is the most common autosomal recessive disease in the world, in the Middle East[1]
We validated a panel of multiplex-PCR based STR genotyping kit (i.e. Hapscreen kit from Genetek Biopharma, Berlin, Germany, GT-500 size standard (GT)) which uses STR markers closely linked to the β-globin gene to aid the diagnose of β-thal as a linkage approach for increasing the accuracy of results obtained by direct method (e.g. Sanger sequencing, MLPA or ARMS methods)
All couples who had applied for Prenatal diagnosis (PND) or preimplantation genetic diagnosis (PGD) were initially visited by a clinical geneticist and their genetic and reproductive history was determined and probable diagnostic choices were discussed

Summary

Introduction

Www.nature.com/scientificreports β-thalassemia (β-thal) is the most common autosomal recessive disease in the world, in the Middle East[1]. Addition of the mentioned markers facilitate aneuploidy screening, detecting maternal cell contamination, confirming fetal sample authenticity in addition to the diagnosis of β-thal simultaneously.

Results

Conclusion