Targeting the Genotype Request 9 th Annual International SADS - - PowerPoint PPT Presentation

Targeting the Genotype Request

9th Annual International SADS Foundation Conference Susan P. Etheridge, MD University of Utah

COI/Declarations

• I had to learn genetics on YouTube to present

this talk

1929 A very long time later, in 1929 ~ 30 years

Sanger Method 1977 (1st generation technology) Human Genome Project 2001 Next-generation sequencing 2005 Clinical application of NGS

Genetic Testing

• Rapidly increasing use in clinical realm
• Identifies affected individuals
• Part of picture

– carriership may not predict clinical outcome – incomplete penetrance and variable expressivity – family members with same mutation, different disease burden

• Genetic and nongenetic factors modify

phenotype

Amin and Wilde J Physiol 2013

Nongenetic Modifiers

Amin and Wilde J Physiol 2013

Genetic Modifiers

Amin and Wilde J Physiol 2013

Targeted panels or whole exome/genome sequencing? How does the physician decide?

“This is a problem for clinicians because they don’t fundamentally understand ………..”

Robert Nussbaum, MD Institute for Human Genetics UCSF

panels broad panels whole exome whole genome

The Beginning

Sequencing - reading through DNA letter by letter

Packaged into strands and wound up for easy storage in nucleus

chromosome gene 4 chemicals combined into a language

is old

Components of Human Genome

Exons

• Protein coding regions of genes
• Make up ~ 1% human genome
• Harbor 85% of mutations with large effects on

disease

• RNA a single strand
• Can travel outside the nucleus Identical to DNA but lacks

intron regions

RNA the recipe Travels to the ribosome – the DNA translating factory Translated into amino acids

synonymous nonsynonymous Single nucleotide change can mean nothing or can mean disease

Sanger sequencing: allowed for sequencing of DNA in a reliable and reproducible manner

Sanger Sequencing

• Used for inherited arrhythmia research and

clinical application

• Gold standard for accuracy
• Useful for hard to target areas
• Validation (exome /genome findings)
• Limited thoughput - slow - 2 million bases/day
• Expensive

We needed something faster

• Whole exome /genome sequencing
• Massively parallel DNA-sequencing ~ 50 billion/day
• Enormous amounts of data cheaply
• Sequence genomes of many organisms
• Enhancing understanding how genetic differences

affect health and disease

SEQUENCING

• Best suited when diagnosis clear (LQTS)
• Reasonable detection rate
• Looking at intronic regions, and now insertions

and deletions

• Chasing a moving target
• rapid new disease genes discovery
• updating and revalidating costly /time-consuming
• how much evidence required to implicate a gene?

panels

Chong Am J Hum Genet. 2015

panels

Many panels available 7-200 genes Panels can be divided into those that are focused (LQTS) and those that are for broader categories of disease (arrhythmia)

broad panels

• Multiple clinical features with no clear diagnosis
• Cases with unusual presentations and no panel available
• Currently available tests very low yield
• Alternative to whole genome sequencing
• Reduced

– costs – turnaround times – data storage needs – informatics burdens

whole exome

After Finding a Mutation

• Go to the literature

– association with disease – functional characterization

• Rarity compared to general population?

(MAF)

• Alteration in protein
• Return to phenotype: present in affected

and absent in controls

Genetic Determination of QT Interval

Rare <1% minor allele frequency (MAF) -large effect on QTc duration – disease causing

‘Common rare’ variants with 1–5% MAF and an intermediate effect on QTc duration Common variants >5% MAF - small effect on QTc duration

• Disease-causing mutations in LQTS families with low

penetrance and variable expressivity

• Variants with strong modifying effects on QTc in

general population

• Variants associated with disease only in co-presence of

a non-genetic trigger (‘second hit’)

– drug-induced LQTS

Priori 1999 Newton-Cheh 2009, Kannankeril 2010

‘Common rare’ variants with 1–5% MAF and an intermediate effect

• n QTc duration

Spectrum of Variants in LQTS

Sauer & Newton-Cheh 2012

Genome Wide Association Study (GWAS)

• DNA extracted from large populations

– with/without QTc prolongation

• Search genome for small variations (SNPs)
• ccurring more in people with QTc

prolongation

• Identify genomic regions that may have not

been previously linked to QTc duration

• No information on cause

Normal QT Long QT

Manhattan Plot

Genetic Architecture of QTc and GWAS

• QT interval duration has a heritable

component

• Genetic factors modify QT duration
• 2006 a signal was identified upstream of

NOS1AP associated with QT interval

• Found to modify LQTS disease severity in

large South African family

Arking Nat Genet 2006, Crotti Circ 2009

QT Interval Associated Loci

COGENT Consortium Nature Genetics 2014

LQTS 1,2,3

LQTS 4-16

Genetically elusive

Conclusions

80,000,000 people in US with cardiovascular disease

• ften heritable

Advanced cutting age genetics/genomics technology perfectly suited to further understanding of inherited arrhythmias and SADs conditions