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Identification of alternative spliced mRNA variants related to cancers by genome-wide ESTs al

ignment

KIM DAE SOO

Oncogene. 2004 Apr

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Alternative splicing Form

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Abstract

Alternative splicing of mRNAs by analysing the exon linkage relationship by alignment of ESTs to the genome sequence

Little effort has been made to investigate the relationship between cancers and alternative splicing

Alternative splicing assembler(ASA) Of 4322 genes screened,3498(81%) were observed

with at least one alternative splicing variants. Using Fisher’s test, alternative splicing variants

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Introduction

About 45% of the human EST sequence are derived from cancer cells.

Alternative splicing is an important mechanism in higher eukaryotes for producing proteomics complexity

Approximately 30-60% genes are alternative splicing as estimated by genomically aligned ESTs

Human could conceivably produce hundreds of thousands of different proteins by the estimated 35,000 genes

Alternative splicing of pre-mRNA is a versatile mechanism for regulating gene function at the post-transcription level

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Alternative splicing defect as indicated by a survey of mutations in splicing junctions.

A total of 26812 alternative spliced variants from 4322 genes were included in the database

2149 variants from 1827 genes were predicted as cancer associated.

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ResultsASA was created to identify alternative splicing of human gene transcripts

52 genes from alternative splicing database ,all having previously reported alternative splicing isoforms.

Only four EST hits were available on the average for these 6 genes ,202 EST hits could be found for the other 46 genes

ASA gives a false negative result of 12 %(about 88% of alternatively splicing genes)

SpliceNest,PALS,HASDB which have false negative rates of 15,35 and 38%

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EST sequences might be contaminated by genomic sequence, vector sequences and chimaeric cDNA clone

Randomly selected 82 splicing variants from 67 genes compared these variants with the NR db

26%(21 out of 82) variants found at least one reported mRNA sequences

selected 13 variants ;RT-PCR (11 of 13 variants were confirmed by RT-PCR)

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Analysis of the alternative spliced variants and their tissue distribution in BASD

A total of 4322 reference sequences were screened and 3490(81%) reference sequences were predicted as alternative spliced ,producing 26,812 splicing variants.

On average six splicing variants were observed for each reference sequences.

87% variants contained less then 16 EST ,and 58% were represented by the EST singletons.

If the singletons were excluded from the database, then only 66% genes were alternative spliced

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A total of 6593 libraries were left and classified into 293 tissue types (NCBI EST data)

A sample of 127 random genes was used to analysis the tissue distribution of alternative splicing

Large fraction of splicing variants are tissue specific

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Identification of cancer-associated splicing variants

about 35% splicing variants were detected exclusively in cancer tissues

29% were only detected in normal tissues This implies that new splicing variants might

be generated during carcinogenesis This result supports the observation that

during carcinogenesis not only is the expression profile affected but the splicing Patten

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This 383 genes of variants will have a relatively higher confidence of cancer relationship.

About 85% cancer related variants had more than 16 ESTs

About 43% of cancer specific variants were related to more than two kinds of cancer types

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Discussion

We constructed BASD to search for splicing variants of gene transcripts especially those associated with cancer

These single EST supported variants in mining genomic information

Our work presents a genomic view of the relationship between cancer and alternative splicing.