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Sequencing, Association Studies ID New Liver Cancer Genes

By a GenomeWeb staff reporter

NEW YORK (GenomeWeb News) – A pair of studies appearing online in Nature Genetics yesterday are providing new clues about the genetic underpinnings and risk factors for a form of liver cancer associated with hepatitis C virus infection.

In the first of these, a team of International Cancer Genome Consortium researchers led by investigators at Japan's National Cancer Center Research Institute sequenced matched tumor and normal genomes from a Japanese man with HCV-induced hepatocellular carcinoma to identify substitutions, rearrangements, and small insertions and deletions in the tumor — information that they validated and expanded upon through whole exome sequencing studies of tumor and normal samples from the same individual.

"This first high-resolution characterization of a virus-associated cancer genome identified previously uncharacterized mutation patterns, intra-chromosomal rearrangements and fusion genes, as well as genetic heterogeneity within the tumor," senior author Tatsuhiro Shibata, a cancer genomics researcher at the National Cancer Center Research Institute, and co-authors wrote.

Using Illumina GAIIx paired-end sequencing, the team sequenced short insert libraries from HCV-positive tumor DNA and matched lymphocyte white blood cell DNA, generating sequence that covered the tumor genome to nearly 36 times coverage and the matched normal genome to just over 28 times coverage.

When they compared the genomes to one another and to the dbSNP database, they found more than 11,700 apparent somatic SNPs in the tumor genome that were absent in the matched normal genome, including an over-representation of non-synonymous substitutions and changes affecting intergenic bits of the tumor genome.

The team found and verified 22 rearrangements in the tumor genome that pointed to specific gene fusions — and found a cluster of rearrangements that grouped together on chromosome 11.

The insertion and deletion patterns detected in the tumor were intriguing as well. Of the 670 small indels they found, researchers explained, seven affected genes. Two of these changes altered known tumor suppressors — TP53 and AXIN1, while five fell in genes linked to other kinds of cancer.

By sequencing the exomes of the tumor and normal samples to about 77 and 74 times coverage, respectively, the researchers tracked down dozens more somatic substitutions. Among them: Sanger sequencing validated alterations in TSC1 — a gene that codes for a component of a protein complex that helps control at least one cancer-related pathway.

"The TSC1 complex, which is inactivated in a sub-population of tumors, negatively regulates the mammalian target of rapamycin signaling, which is an important oncogenic pathway related the growth, metabolism, and stemness of cancer cells," the team concluded, "and could be a promising molecular therapeutic target in [hepatocellular carcinoma] progression."

Meanwhile, through a genome-wide association study involving 721 individuals with HCV-associated liver cancer and 2,890 unaffected controls from Japan, researchers from the University of Tokyo and RIKEN initially found a handful of liver cancer linked SNPs.

In their subsequent replication experiments involving thousands more individuals with or without HCV-associated liver cancer, the team narrowed these down to a lone risk locus near the chromosome 6 gene MICA.

While their follow-up experiments didn't show any ties between this SNP and risk of chronic hepatitis C infection, the team did find clues suggesting the variant influences the risk that such an infection will progress to liver cancer.

"Although the molecular mechanism whereby MICA polymorphisms confer the risk of disease progression should be characterized in the future," senior author Koichi Matsuda, a researcher with the University of Tokyo's Human Genome Center, and co-authors wrote, "our findings reveal a crucial role of genetic variations in the host innate immune system in the development of HCV-induced [hepatocellular carcinoma]."

Matsuda and his co-workers initially used Illumina HumanHap610-Quad and HumanHap550v3 Genotyping BeadChip arrays to genotyped affected and unaffected participants at more than 430,000 SNPs across the genome, identifying eight potential risk SNPs.

When they tested these SNPs in another 673 individuals with HCV-induced liver cancer and 2,596 individuals without HCV, the team found just one SNP that remained significantly associated with the disease: the chromosome 6 SNP rs2596542, found in major histocompatibility complex region upstream of MICA.

The variant didn't associate with chronic hepatitis C infection risk, researchers found when they looked at 1,730 infected individuals who had not yet developed complications such as cirrhosis or liver cancer.

Instead, they explained, their follow-up experiments hint that the SNP is tied to both muted MICA expression levels and enhanced risk of transitioning from long-term hepatitis infection to liver cancer.

As such, those involved in the new GWAS argue that their findings may underscore to a broader role for host immune function in liver cancer risk.