Genetic Cancer Susceptibility (GCS)

Genomics in I4C

James McKay

GCS aims and focus

I. Identify and understand genetic susceptibility to cancer

• applying cutting edge genomics techniques to the large bio-repositories within GEN

• lung, head and neck, kidney cancers and lymphomas

II. Genetics Platform (GSP) • Develop and maintain genotyping / genomic

applications

• Including the related bioinformatics

GCS studiesCancers prioritized by IARC

GWAS – lung, hodgkin’s lymphoma, UADT cancers

Pedigree analysis – Exome sequencing in unusual pedigrees

NPC cancers in Sarawak, Malaysia,

Bladder Cancer in Iran

Novel strategies. Use an intregrative approach to maximise our ability to perform meaningful studies within limited sample sets

(mutation profiles, loss of second copy of alleles. eQTL)

Bidayuh NPC pedigreeKuching Sarawak Malaysia

Pedigree in a loose sense, three isolated villages

Discovery Exome sequencing + linkage analysis (snp arrays) Assess for segregation

Validation Screening candidate genes in additional cases

Genetics platform Flexibility, suitability to IARC studies and

bioropositories• Next generation sequencing LifeTechnology 5500 and PGM

• Illumina bead array

• High performance computer cluster

• Highly dynamic field, what possible this week, needs to be revised next week

Rapid and cheap comprehensive sequencing of many tumours is only now becoming feasible

72.8 €/sample

12.2 €/sample

X 6 cost through GCS optimization

Targeted Resequencing Ion Torrent Experiment Cost (35 amplicons, 150X coverage)

Exome/Genome SequencingMany genes/Few samples

Targeted ResequencingFew genes/Many samples

Genes mutated within HNC

Stransky N et al (2011). Science

• Within 100 (TCGA) head and neck tumours, 95% mutations reported within 10 genes.

• It becomes possible to design targeted sequencing assays to cover most mutations, and at low cost.

• Mutations can be detected in the DNA from plasma (0.1-1%)

Highly multiplexed PCR used for enrichment.

Targeted combined amplification of genes

up to 300-400 amplicons/PCR5 genes>240,000 ampliconswhole exomes

Using this to resequence candidate genes. 20 euros/sequence five genes.

Applied to circulate tumour DNA (10,000X) to capture rare events

Qaigen Generead

I4C genomics applicationsTEL/AML translocations

What is there prevalence at birth (1% or 0.1%?). Dosage?

Is presence of TEL/AML translocations predictive of ALL later in life?

Is there a relationship between TEL/AML dose/patient and ALL?

Is modified by other factors? Birthweight, age of onset (early ALL cases), gender…

TEL/AML translocationassayed using RNA

Limitation is the availability of appropriate bio-specimens

A Zelent et al, Oncogene (2004)

DNA assay for TEL/AML translocationComplicated as the breakpoint for the translocations is the variable.

TEL (ETV6) gene

12 kb

AML1 (RUNX1) gene

100 kb

Tiled highly multiplexed PCR

Low strigency PCR, coupled with very deep sequencing (10,000) to capture very rare variants

N=60 (200bp)

N=500 (200bp)