Long-read sequencing identified a causal structural variant in an exome-negative case and enabled preimplantation genetic diagnosis

Hefan Miao,Bodi Gao,Fan Liang,Qianjun Zhang,Depeng Wang,Juan Du,Qi Yang,Kai Wang,Na Ma,Ge Lin,Jiapeng Zhou

doi:10.1186/s41065-018-0069-1

Abstract

BackgroundFor a proportion of individuals judged clinically to have a recessive Mendelian disease, only one heterozygous pathogenic variant can be found from clinical whole exome sequencing (WES), posing a challenge to genetic diagnosis and genetic counseling. One possible reason is the limited ability to detect disease causal structural variants (SVs) from short reads sequencing technologies. Long reads sequencing can produce longer reads (typically 1000 bp or longer), therefore offering greatly improved ability to detect SVs that may be missed by short-read sequencing.ResultsHere we describe a case study, where WES identified only one heterozygous pathogenic variant for an individual suspected to have glycogen storage disease type Ia (GSD-Ia), which is an autosomal recessive disease caused by bi-allelic mutations in the G6PC gene. Through Nanopore long-read whole-genome sequencing, we identified a 7.1 kb deletion covering two exons on the other allele, suggesting that complex structural variants (SVs) may explain a fraction of cases when the second pathogenic allele is missing from WES on recessive diseases. Both breakpoints of the deletion are within Alu elements, and we designed Sanger sequencing and quantitative PCR assays based on the breakpoints for preimplantation genetic diagnosis (PGD) for the family planning on another child. Four embryos were obtained after in vitro fertilization (IVF), and an embryo without deletion in G6PC was transplanted after PGD and was confirmed by prenatal diagnosis, postnatal diagnosis, and subsequent lack of disease symptoms after birth.ConclusionsIn summary, we present one of the first examples of using long-read sequencing to identify causal yet complex SVs in exome-negative patients, which subsequently enabled successful personalized PGD.

Highlights

For a proportion of individuals judged clinically to have a recessive Mendelian disease, only one heterozygous pathogenic variant can be found from clinical whole exome sequencing (WES), posing a challenge to genetic diagnosis and genetic counseling
Several published studies demonstrated examples where disease causal structural variants (SVs) were missed by short-read WES/whole-genome sequencing (WGS) [16], or that certain classes of disease causal repeats failed to be identified by WES/WGS [17]
Clinical examination We were presented with a 12 year-old boy with hepatosplenomegaly and growth retardation at the Xiangya Hospital of Central South University, Hunan, China in 2017 (Fig. 1a)

Summary

Introduction

For a proportion of individuals judged clinically to have a recessive Mendelian disease, only one heterozygous pathogenic variant can be found from clinical whole exome sequencing (WES), posing a challenge to genetic diagnosis and genetic counseling. Miao et al Hereditas (2018) 155:32 variants, the possibility of complicated oligogenic disease in certain patients and the possibility of disease causal mechanisms due to somatic or epigenetic origin Among these reasons, the limited ability to interrogate repeat elements such as tandem repeats [9] and structural variants (SVs) [10] may play important roles. The limited ability to interrogate repeat elements such as tandem repeats [9] and structural variants (SVs) [10] may play important roles Clinical microarrays such as SNP arrays or array-CGH can detect relatively large deletions and duplications [11,12,13,14], but have difficulty to reliably identify small (< 10 kb) exonic deletions or complex SVs in clinical settings [15]. Conventional short-reads sequencing approaches have been reported to lack sensitivity, exhibit very high false positive rate, and misinterpret complex or nested SVs [18]

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Conclusion