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Advances in genetic technologies in the identification of genetic disease
in children
Dr Katie SnapeSpecialist 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 Smaller scale
Aneuploidy Structural rearrangements
Base substitutions
Small insertions and deletions
Genetic variation
Large scale Smaller scale
Aneuploidy Structural rearrangements
Base substitutions
Small insertions and deletions
Single Nucleotide Polymorphism (SNP)
Genetic variation
Large scale Smaller scale
Aneuploidy Structural rearrangements
Base substitutions
Small insertions and deletions
Genetic variation
Large scale Smaller scale
Aneuploidy Structural rearrangements
Small insertions and deletions
CYTOGENETIC ANALYSIS DNA SEQUENCING
Base substitutions
Genetic variation
Large scale Smaller scale
Aneuploidy Structural rearrangements
Small insertions and deletions
CYTOGENETIC ANALYSIS DNA SEQUENCING
Base substitutions
Cytogenetic analysis• What used to happen…..
Fluorescent In-Situ Hybridisation
Developmental delay
Congenital heart disease
Hypocalcaemia
Array CGH
• An array is a glass slide onto which thousands of short sequences of DNA (probes) are spotted.
AND NOW….
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
Large scale Smaller scale
Aneuploidy Structural rearrangements
Small insertions and deletions
CYTOGENETIC ANALYSIS DNA SEQUENCING
Base substitutions
DNA sequencingGenomic 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”
• The coding part of ~ 20000 genes• Most likely to harbour disease causing
mutations
1 Gene
Data Analysis
• 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?
• 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
