Advances in genetic technologies in the identification of genetic disease in children Dr Katie Snape Specialist Registrar in Genetics St Georges Hospital.

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