Advances in genetic technologies in
the identification of genetic disease
in children
Dr Katie Snape
Specialist Registrar in Genetics
St Georges Hospital
DNA and the genetic code
• Made up of 4
nucleotides or “bases”
–
–
–
–
A = Adenine
T = Thymine
C = Cytosine
G = Guanine
5’-ATGTGCATGCTAGCT-3’
3’-TACACGTACGATCGA-5’
Genetic variation
• Makes us unique
– “polymorphisms”
• Is the basis for
evolution
• Is the basis for disease
http://dee-annarogers.com
Genetic variation
Large scale
Genetic variation
Large scale
Aneuploidy
Genetic variation
Large scale
Aneuploidy
Structural
rearrangements
Genetic variation
Large scale
Aneuploidy
Structural
rearrangements
Smaller scale
Base
substitutions
Small insertions
and deletions
Genetic variation
Large scale
Aneuploidy
Structural
rearrangements
Smaller scale
Base
substitutions
Small insertions
and deletions
Single Nucleotide
Polymorphism (SNP)
Genetic variation
Large scale
Aneuploidy
Structural
rearrangements
Smaller scale
Base
substitutions
Small insertions
and deletions
Genetic variation
CYTOGENETIC ANALYSIS
Large scale
Aneuploidy
Structural
rearrangements
DNA SEQUENCING
Smaller scale
Base
substitutions
Small insertions
and deletions
Genetic variation
CYTOGENETIC ANALYSIS
Large scale
Aneuploidy
Structural
rearrangements
DNA SEQUENCING
Smaller scale
Base
substitutions
Small insertions
and deletions
Cytogenetic analysis
• What used to happen…..
Fluorescent In-Situ Hybridisation
Developmental delay
Congenital heart disease
Hypocalcaemia
AND NOW….
Array CGH
• An array is a glass slide onto which
thousands of short sequences of DNA
(probes) are spotted.
Array CGH
Array CGH
Submicroscopic chromosomal
abnormalities
• Contiguous gene syndromes
– Phenotype conferred by haploinsufficiency or gain
of multiple different genes
• Common clinical features
– Developmental delay
– Facial dysmorphism
– Congenital abnormalities
Interpretation
• Copy number variant vs pathogenic mutation
• Parental studies – is variant de novo?
– Caution!
• Is parent also affected?
• Is the phenotype variable?
• Genetic material in region
– Does gain or loss of genes match phenotype?
• Comparison with other children
– Decipher database
Array CGH
• Making more diagnoses than ever before
but…
– Can lead to clinical uncertainty
– Do not over interpret array findings
– Remember WE ARE ALL INDIVIDUALS
Genetic variation
CYTOGENETIC ANALYSIS
Large scale
Aneuploidy
Structural
rearrangements
DNA SEQUENCING
Smaller scale
Base
substitutions
Small insertions
and deletions
DNA sequencing
Genomic DNA
Primer amplification
of region of interest
Cycle sequencing
with fluorescently
labelled chain
terminator ddNTPs
Capillary
Electrophoresis
(1 read/capillary)
Sanger sequencing
• 500-600bp per reaction
• Takes > 1 year to sequence 1 gigabase (1/3 of
human genome)
• Costs $0.10 per 1000 bases
• The Human Genome Project took >10 years
• And now…..
Next Generation Sequencing (NGS)
• Multiple methodological approaches
• In practice….
– Single molecule sequencing
– Massively parallel sequencing
• Whole genome sequencing – in a week
• Targeted resequencing
– “exome”
Single-molecule sequencing
Massively parallel sequencing
Fragment DNA
Fragment DNA
Amplify DNA fragments of interest
Fragment DNA
Amplify DNA fragments of interest
Sequence DNA fragments in parallel
Fragment DNA
Amplify DNA fragments of interest
Sequence DNA fragments in parallel
Generate data containing 100 bp DNA reads
Fragment DNA
Amplify DNA fragments of interest
Sequence DNA fragments in parallel
Generate data containing 100 bp DNA reads
Align DNA reads to reference genome
Fragment DNA
Amplify DNA fragments of interest
Sequence DNA fragments in parallel
Generate data containing 100 bp DNA reads
Align DNA reads to reference genome
Identify differences between sample and reference
“Variant calling”
The “Exome”
1 Gene
• The coding part of ~ 20000 genes
• Most likely to harbour disease causing
mutations
Data Analysis
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•
•
•
15-20 Gb of data per exome stored
Files contain sequence reads of ~100 bases
Need to align reads to reference genome
Need to call variants seen in an individual
sample
Alignment
Variant calling
• Reads = the strands of DNA which are aligned
with the reference sequence
• Depth of coverage = number of reads covering
a particular region of the exome
– The deeper the coverage, the more accurate the
results
– Alterations within the middle of a read are more
likely real than those at the end of a read
Variant calling
Clinical Applications
• Identification of novel disease genes in
Mendelian disorders
• Identification of genetic susceptibility to
common and complex disorders
• Rapid sequencing of multiple known genes
– Diagnostic gene panels
• Guide therapeutics
– Sequencing of cancer genomes
– Pharmacogenetics
Identifying Mendelian disease genes
• Per genome ~ 3 million variants per sample
• Per exome ~ 20, 000 variants per sample
– How can we go from 20, 000 to 1?
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•
•
•
Genes shared in multiple affected individuals
Inheritance patterns in a family
Look for RARE genetic variants
De novo variants
Diagnostic gene panels
• Genetically heterogenous
disorders
– Previously, sequential
sequencing of genes
– Time consuming and expensive
• NGS allows all known genes
to be sequenced in parallel
e.g For Noonan syndrome
• PTPN11, SOS1, RAF1, KRAS, NRAS, BRAF,
MEK1, MEK2, HRAS, SHOC2, CBL,
SPRED1
Pitfalls
• Variants of uncertain clinical significance
• Incidental findings e.g mutations in genes for
adult onset conditions
Conclusions
• Unprecedented opportunities to identify
genetic factors influencing disease
• Genetic technologies will become
commonplace in diagnostics and therapeutics
• Array CGH and NGS likely to become first line
diagnostic testing techniques in clinical
paediatrics
• We should be cautious of over interpretation
of genetic data