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Whole-exome sequencing of fibroblast and its iPS cell lines derived from a patient diagnosed with xeroderma pigmentosum.

Okamura K, Toyoda M, Hata K, Nakabayashi K, Umezawa A - Genom Data (2015)

Bottom Line: Unexpectedly, we failed to find causal mutations in the XP-related genes, but we identified many other mutations including homozygous deletion of GSTM1 and GSTT1.In addition, we found that the long arm of chromosome 9 formed uniparental disomy in the iPS cell line, which was also confirmed by a structural mutation analysis using a SNP array.Taken together, we conclude that the patient might be affected by a different type of the disorder and that some of the mutations that we identified here may be responsible for exhibiting the phenotype.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems BioMedicine, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan.

ABSTRACT
Cells from a patient with a DNA repair-deficiency disorder are anticipated to bear a large number of somatic mutations. Because such mutations occur independently in each cell, there is a high degree of mosaicism in patients' tissues. While major mutations that have been expanded in many cognate cells are readily detected by sequencing, minor ones are overlaid with a large depth of non-mutated alleles and are not detected. However, cell cloning enables us to observe such cryptic mutations as well as major mutations. In the present study, we focused on a fibroblastic cell line that is derived from a patient diagnosed with xeroderma pigmentosum (XP), which is an autosomal recessive disorder caused by a deficiency in nucleotide excision repair. By making a list of somatic mutations, we can expect to see a characteristic pattern of mutations caused by the hereditary disorder. We cloned a cell by generating an iPS cell line and performed a whole-exome sequencing analysis of the progenitor and its iPS cell lines. Unexpectedly, we failed to find causal mutations in the XP-related genes, but we identified many other mutations including homozygous deletion of GSTM1 and GSTT1. In addition, we found that the long arm of chromosome 9 formed uniparental disomy in the iPS cell line, which was also confirmed by a structural mutation analysis using a SNP array. Type and number of somatic mutations were different from those observed in XP patients. Taken together, we conclude that the patient might be affected by a different type of the disorder and that some of the mutations that we identified here may be responsible for exhibiting the phenotype. Sequencing and SNP-array data have been submitted to SRA and GEO under accession numbers SRP059858 and GSE55520, respectively.

No MeSH data available.


Related in: MedlinePlus

Genome-wide distribution of the somatic mutations identified in the present study.Blue and red bars indicate single-nucleotide and indel mutations, respectively. Nearly one thousand mutations were detected on the long arm of chromosome 9. The discrepant genotypes between the progenitor and its iPS cell lines were caused by a single structural mutation that formed uniparental disomy in the chromosome arm.
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f0005: Genome-wide distribution of the somatic mutations identified in the present study.Blue and red bars indicate single-nucleotide and indel mutations, respectively. Nearly one thousand mutations were detected on the long arm of chromosome 9. The discrepant genotypes between the progenitor and its iPS cell lines were caused by a single structural mutation that formed uniparental disomy in the chromosome arm.

Mentions: Next, we searched for inconsistent genotypes called with UnifiedGenotyper between the progenitor and its iPS cell lines to identify somatic mutations. Using the criteria described in our previous study [6], 922 single-nucleotide substitutions and 77 indels were identified. Because the numbers were extremely high compared to those of the preceding studies, we examined the positions of all the genotypes that exhibited disagreement between the two lines (Fig. 1). Among these sites, 845 single-nucleotide and 69 indel sites were located on the long arm of chromosome 9. All the called genotypes were homozygous without exception. Using an Illumina HumanCytoSNP-12 v2.1 DNA Analysis BeadChip Kit, we also performed a SNP-genotyping analysis to examine the existence of structural alterations. Copy-neutral loss of heterozygosity was detected in the 9:71,035,938–138,893,874 region (GRCh37) of the iPS cell line, indicating that a large structural mutation formed uniparental disomy. In that case, heterozygous sites in the region became homozygous. The 914 sites in the exome-target regions were considered to be just heterozygous in the progenitor cell, but not individual mutations. The SNP-genotyping analysis also suggested one copy-gain mutation around 16:83,939,438–85,567,156 only in the iPS cell line, but no genotype disagreement was found in this 1.6-Mb region. Among the remaining 77 single-nucleotide substitutions, C-to-T or G-to-A transitions accounted for 29 of the changes.


Whole-exome sequencing of fibroblast and its iPS cell lines derived from a patient diagnosed with xeroderma pigmentosum.

Okamura K, Toyoda M, Hata K, Nakabayashi K, Umezawa A - Genom Data (2015)

Genome-wide distribution of the somatic mutations identified in the present study.Blue and red bars indicate single-nucleotide and indel mutations, respectively. Nearly one thousand mutations were detected on the long arm of chromosome 9. The discrepant genotypes between the progenitor and its iPS cell lines were caused by a single structural mutation that formed uniparental disomy in the chromosome arm.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664661&req=5

f0005: Genome-wide distribution of the somatic mutations identified in the present study.Blue and red bars indicate single-nucleotide and indel mutations, respectively. Nearly one thousand mutations were detected on the long arm of chromosome 9. The discrepant genotypes between the progenitor and its iPS cell lines were caused by a single structural mutation that formed uniparental disomy in the chromosome arm.
Mentions: Next, we searched for inconsistent genotypes called with UnifiedGenotyper between the progenitor and its iPS cell lines to identify somatic mutations. Using the criteria described in our previous study [6], 922 single-nucleotide substitutions and 77 indels were identified. Because the numbers were extremely high compared to those of the preceding studies, we examined the positions of all the genotypes that exhibited disagreement between the two lines (Fig. 1). Among these sites, 845 single-nucleotide and 69 indel sites were located on the long arm of chromosome 9. All the called genotypes were homozygous without exception. Using an Illumina HumanCytoSNP-12 v2.1 DNA Analysis BeadChip Kit, we also performed a SNP-genotyping analysis to examine the existence of structural alterations. Copy-neutral loss of heterozygosity was detected in the 9:71,035,938–138,893,874 region (GRCh37) of the iPS cell line, indicating that a large structural mutation formed uniparental disomy. In that case, heterozygous sites in the region became homozygous. The 914 sites in the exome-target regions were considered to be just heterozygous in the progenitor cell, but not individual mutations. The SNP-genotyping analysis also suggested one copy-gain mutation around 16:83,939,438–85,567,156 only in the iPS cell line, but no genotype disagreement was found in this 1.6-Mb region. Among the remaining 77 single-nucleotide substitutions, C-to-T or G-to-A transitions accounted for 29 of the changes.

Bottom Line: Unexpectedly, we failed to find causal mutations in the XP-related genes, but we identified many other mutations including homozygous deletion of GSTM1 and GSTT1.In addition, we found that the long arm of chromosome 9 formed uniparental disomy in the iPS cell line, which was also confirmed by a structural mutation analysis using a SNP array.Taken together, we conclude that the patient might be affected by a different type of the disorder and that some of the mutations that we identified here may be responsible for exhibiting the phenotype.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems BioMedicine, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan.

ABSTRACT
Cells from a patient with a DNA repair-deficiency disorder are anticipated to bear a large number of somatic mutations. Because such mutations occur independently in each cell, there is a high degree of mosaicism in patients' tissues. While major mutations that have been expanded in many cognate cells are readily detected by sequencing, minor ones are overlaid with a large depth of non-mutated alleles and are not detected. However, cell cloning enables us to observe such cryptic mutations as well as major mutations. In the present study, we focused on a fibroblastic cell line that is derived from a patient diagnosed with xeroderma pigmentosum (XP), which is an autosomal recessive disorder caused by a deficiency in nucleotide excision repair. By making a list of somatic mutations, we can expect to see a characteristic pattern of mutations caused by the hereditary disorder. We cloned a cell by generating an iPS cell line and performed a whole-exome sequencing analysis of the progenitor and its iPS cell lines. Unexpectedly, we failed to find causal mutations in the XP-related genes, but we identified many other mutations including homozygous deletion of GSTM1 and GSTT1. In addition, we found that the long arm of chromosome 9 formed uniparental disomy in the iPS cell line, which was also confirmed by a structural mutation analysis using a SNP array. Type and number of somatic mutations were different from those observed in XP patients. Taken together, we conclude that the patient might be affected by a different type of the disorder and that some of the mutations that we identified here may be responsible for exhibiting the phenotype. Sequencing and SNP-array data have been submitted to SRA and GEO under accession numbers SRP059858 and GSE55520, respectively.

No MeSH data available.


Related in: MedlinePlus