Molecular characterization of ring chromosome 18 by low-coverage next generation sequencing

Abstract

Background: Ring chromosomes are one category of structurally abnormal chromosomes that can lead to severe growth retardation and other clinical defects. Traditionally, their diagnosis and characterization has largely relied on conventional cytogenetics and fluorescence in situ hybridization, array-based comparative genomic hybridization and single nucleotide polymorphism array-based comparative genomic hybridization. However, these methods are ineffectively at characterizing the ring chromosome structure and only offer a low resolution mapping of breakpoints. Here, we applied whole-genome low-coverage paired-end next generation sequencing (NGS) to two suspected cases of ring chromosome 18 (r(18)) and characterized the ring structure including the chromosome dosage changes and the breakpoint junction.

Methods: The breakpoints and chromosome copy number variations (CNVs) of r(18) were characterized by whole-genome low-coverage paired-end NGS. We confirmed the dosage change by single nucleotide polymorphisms array, and validated the junction site regions using PCR followed by Sanger sequencing.

Results: We successfully and fully characterized the r(18) in two cases by NGS. We mapped the breakpoints with a high resolution and identified all CNVs in both cases. We analyzed the breakpoint regions and discovered two breakpoints located within repetitive sequence regions, and two near the repetitive sequence regions. One of the breakpoints in case 2 was located within the gene METTL4, while the other breakpoints were intergenic.

Conclusions: We demonstrated that whole-genome low-coverage paired-end NGS can be used directly to map breakpoints with a high molecular resolution and detect all CNVs on r(18). This approach will provide new insights into the genotype-phenotype correlations on r(18) and the underlying mechanism of ring chromosomes formation. Our results also demonstrate that this can be a powerful approach for the diagnosis and characterization of ring chromosomes in the clinic.

Fig. 1

Deletion on both ends of the chromosome 18. a Karyotypes of chromosome 18 in both cases. The normal chromosome is shown on the left and the abnormal one on the right. b CNV detected by NGS. c CNV detected by the SNP array

Fig. 2

Characterization of r(18) and mapping of the breakpoints. a Schematic of chimeric mate-pair reads on chromosome 18 spanning the putative junction site (JS) in both cases. b Junction site sequences amplified by PCR (left, L1) and breakpoints (arrows) defined by Sanger sequencing (right). Genomic DNA from healthy individual was used as a negative control (left, L2). Two nucleotide variations on junction fragments in case 2 are marked in lower case and asterisked

Fig. 3

Chromosome breakpoints and disrupted genes. Breakpoints are indicated by red and green arrows. No genes were disrupted by any of the breakpoints in case 1 or by one of the breakpoints in case 2. The second breakpoint in case 2 on the long arm of chromosome 18 disrupted METTL4