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

Ji X, Liang D, Sun R, Liu C, Ma D, Wang Y, Hu P, Xu Z - BMC Med. Genet. (2015)

Bottom Line: However, these methods are ineffectively at characterizing the ring chromosome structure and only offer a low resolution mapping of breakpoints.We successfully and fully characterized the r(18) in two cases by NGS.Our results also demonstrate that this can be a powerful approach for the diagnosis and characterization of ring chromosomes in the clinic.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Reproductive Medicine, Department of Prenatal Diagnosis, Nanjing Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing, China. xji0501@163.com.

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.

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Related in: MedlinePlus

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
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Fig2: 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

Mentions: Based on the NGS data, we analyzed chimeric mate-pair reads with both ends mapping to different genomic regions. We detected four and five chimeric mate-pairs spanning the putative junction sites of chromosome 18 in case 1 and case 2, respectively (Fig. 2a, Table 1). On the basis of their position, the breakpoints were estimated to be located 5' to chr18: 11172407 and 3' to chr18: 58608193 in case 1 and 5' to chr18: 2551851 and 3' to chr18: 63115329 in case 2.Fig. 2


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

Ji X, Liang D, Sun R, Liu C, Ma D, Wang Y, Hu P, Xu Z - BMC Med. Genet. (2015)

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
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4557216&req=5

Fig2: 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
Mentions: Based on the NGS data, we analyzed chimeric mate-pair reads with both ends mapping to different genomic regions. We detected four and five chimeric mate-pairs spanning the putative junction sites of chromosome 18 in case 1 and case 2, respectively (Fig. 2a, Table 1). On the basis of their position, the breakpoints were estimated to be located 5' to chr18: 11172407 and 3' to chr18: 58608193 in case 1 and 5' to chr18: 2551851 and 3' to chr18: 63115329 in case 2.Fig. 2

Bottom Line: However, these methods are ineffectively at characterizing the ring chromosome structure and only offer a low resolution mapping of breakpoints.We successfully and fully characterized the r(18) in two cases by NGS.Our results also demonstrate that this can be a powerful approach for the diagnosis and characterization of ring chromosomes in the clinic.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Reproductive Medicine, Department of Prenatal Diagnosis, Nanjing Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing, China. xji0501@163.com.

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.

Show MeSH
Related in: MedlinePlus