Haplotyping by linked-read sequencing (HLRS) of the genetic disease carriers for preimplantation genetic testing without a proband or relatives

Qing Li,Hongzi Du,Bo Liang,Jianqiao Liu,Wenzhi He,Shaoying Li,Yan Mao,Lingyin Kong,Jianchun He,Jun Zhang

doi:10.1186/s12920-020-00766-1

Abstract

BackgroundIn order to mitigate the risk of allele dropout (ADO) and ensure the accuracy of preimplantation genetic testing for monogenic disease (PGT-M), it is necessary to construct parental haplotypes. Typically, haplotype resolution is obtained by genotyping multiple polymorphic markers in both parents and a proband or a relative. Sometimes, single sperm typing, or tests on the polar bodies may also be useful. Nevertheless, this process is time-consuming. At present, there was no simple linkage analysis strategy for patients without affected relatives.MethodTo solve this problem, we established a haplotyping by linked-read sequencing (HLRS) method without the requirement for additional relatives. First, the haplotype of the genetic disease carriers in the family was constructed by linked-read sequencing, and then the informative single nucleotide polymorphisms (SNPs) in upstream and downstream mutation region were selected to construct the embryo haplotype and to determine whether the embryo was carrying the mutation. Two families were selected to validate this method; one with alpha thalassemia and the other with NDP gene disorder.ResultsThe haplotyping by linked-read sequencing (HLRS) method was successfully applied to construct parental haplotypes without recruiting additional family members; the method was also validated for PGT-M. The mutation carriers in these families were sequenced by linked-read sequencing, and their haplotypes were successfully phased. Adjacent SNPs of the mutation gene were identified. The informative SNPs were chosen for linkage analyses to identify the carrier embryos. For the alpha thalassemia family, a normal blastocyst was transferred to the uterus and the accuracy of PGT-M was confirmed by amniocentesis at 16 weeks of gestation.ConclusionsOur results suggest that HLRS can be applied for PGT-M of monogenic disorders or de novo mutations where the mutations haplotype cannot be determined due to absence of affected relatives.

Highlights

In order to mitigate the risk of allele dropout (ADO) and ensure the accuracy of preimplantation genetic testing for monogenic disease (PGT-M), it is necessary to construct parental haplotypes
For the alpha thalassemia family, a normal blastocyst was transferred to the uterus and the accuracy of PGT-M was confirmed by amniocentesis at 16 weeks of gestation
Our results suggest that haplotyping by linked-read sequencing (HLRS) can be applied for PGT-M of monogenic disorders or de novo mutations where the mutations haplotype cannot be determined due to absence of affected relatives

Summary

Introduction

In order to mitigate the risk of allele dropout (ADO) and ensure the accuracy of preimplantation genetic testing for monogenic disease (PGT-M), it is necessary to construct parental haplotypes. Single sperm typing, or tests on the polar bodies may be useful. Preimplantation genetic testing for monogenic disease (PGT-M) is a clinical diagnostic procedure that can effectively prevent the implantation of embryos with genetic defects, without the need of gamete donation or adoption [1, 2]. ADO refers to the failure of one of the two alleles of a heterozygous locus This makes a heterozygous cell appear homozygous at the affected locus, leading to misdiagnosis. The affected and unaffected embryos are distinguished using haplotype-phasing results, where the familial linkage analyses are performed using both the couple’s and a proband’s or relatives’ genome

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Conclusion