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

2. 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’

4. Genetic variation Makes us unique Is the basis for evolution
“polymorphisms”
Is the basis for evolution
Is the basis for disease

5. Genetic variation
Large scale

6. Genetic variation
Large scale
Aneuploidy

7. Structural rearrangements
Genetic variation
Large scale
Aneuploidy
Structural rearrangements

8. Structural rearrangements Small insertions and deletions
Genetic variation
Large scale
Smaller scale
Aneuploidy
Structural rearrangements
Base substitutions
Small insertions and deletions

9. Structural rearrangements Small insertions and deletions
Genetic variation
Large scale
Smaller scale
Aneuploidy
Structural rearrangements
Base substitutions
Small insertions and deletions
Single Nucleotide Polymorphism (SNP)

10. Structural rearrangements Small insertions and deletions
Genetic variation
Large scale
Smaller scale
Aneuploidy
Structural rearrangements
Base substitutions
Small insertions and deletions

11. Structural rearrangements Small insertions and deletions
Genetic variation
CYTOGENETIC ANALYSIS
DNA SEQUENCING
Large scale
Smaller scale
Aneuploidy
Structural rearrangements
Base substitutions
Small insertions and deletions

12. Structural rearrangements Small insertions and deletions
Genetic variation
CYTOGENETIC ANALYSIS
DNA SEQUENCING
Large scale
Smaller scale
Aneuploidy
Structural rearrangements
Base substitutions
Small insertions and deletions

13. Cytogenetic analysis
What used to happen…..

17. Fluorescent In-Situ Hybridisation
Developmental delay
Congenital heart disease
Hypocalcaemia

18. AND NOW….
Array CGH
An array is a glass slide onto which thousands of short sequences of DNA (probes) are spotted.

22. Array CGH

23. Array CGH

24. 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

25. 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

27. 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

28. Structural rearrangements Small insertions and deletions
Genetic variation
CYTOGENETIC ANALYSIS
DNA SEQUENCING
Large scale
Smaller scale
Aneuploidy
Structural rearrangements
Base substitutions
Small insertions and deletions

29. DNA sequencing Genomic DNA Primer amplification of region of interest
Cycle sequencing with fluorescently labelled chain terminator ddNTPs
Capillary Electrophoresis
(1 read/capillary)

30. 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…..

31. Next Generation Sequencing (NGS)
Multiple methodological approaches
In practice….
Single molecule sequencing
Massively parallel sequencing
Whole genome sequencing – in a week
Targeted resequencing
“exome”

32. Single-molecule sequencing

33. Massively parallel sequencing

34. Fragment DNA

35. Amplify DNA fragments of interest
Fragment DNA
Amplify DNA fragments of interest

36. Amplify DNA fragments of interest
Fragment DNA
Amplify DNA fragments of interest
Sequence DNA fragments in parallel

37. Amplify DNA fragments of interest
Fragment DNA
Amplify DNA fragments of interest
Sequence DNA fragments in parallel
Generate data containing 100 bp DNA reads

38. Amplify DNA fragments of interest
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

39. Amplify DNA fragments of interest
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”

40. The “Exome” The coding part of ~ 20000 genes
Most likely to harbour disease causing mutations

41. 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

42. Alignment

43. 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

44. Variant calling

45. 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

46. 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

47. 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

48. Pitfalls Variants of uncertain clinical significance
Incidental findings e.g mutations in genes for adult onset conditions

49. 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