Developments in Laboratory Genetics Rachel Butler rachel.butler@wales.nhs.uk The Impact of Genomics on Public Health
Where are we now? Single gene disorders Core disorders Newborn screening (CF, MCAD) UKGTN for rare(r) disorders 695 gene / disease combinations (+67)
Where are we now? Single gene disorders Antenatal screening by QF-PCR D21S1437 3 alleles D21S11 D13S628 D13S634 D18S535 D18S391 D13S742 D18S386 D21S1413 D21S1411
Where are we now? Single gene disorders Antenatal screening by QF-PCR Arrays for LD
Where are we going? Free-fetal DNA Next generation sequencing Personalised medicine
Free fetal DNA (ffdna) Applications Sexing Rhesus D Single gene disorders Aneuploidy analysis (RAPID)
Extraction of cell free fetal nucleic acids from maternal plasma Cell free maternal DNA (96.6%) Cell free fetal DNA (3.4%) Plasma Amount of cell free fetal DNA extracted is equivalent to 25 cells / ml plasma Cell free maternal RNA Cell free fetal RNA Maternal blood cells
Detecting ffdna in maternal blood.
Taken from Cell free fetal nucleic acids for non-invasive prenatal diagnosis; PHGF, 2009
Fetal sexing Taken from CMGS Audit 2011-12
Single gene disorders DF508 / N G551D / N N / N N / Exp ffdna PND DF508 / N N / N G551D / N G551D / DF508 Cystic fibrosis PND N / N N / Exp Huntingtons
Single gene disorders Services now available for achondroplasia and thanatophoric dysplasia Others being piloted through RAPID for: Aicardi Goutiere, Alagille, Barth, Cockayne, Craniosynostoses, Dominant Ataxias, Ectodermal dysplasia, Ellis van Creveld, Fragile X, Friedrich s ataxia, Gylcogen storage diseases, Huntington s, Holt Oram, Inborn errors of metabolism, IPEX, Lissencephaly, Meckel gruber, Myotonic dystrophy, Neonatal diabetes/familial hyperinsulinaemias, Neurofibromatosis ½, Okihiro, Ornthine transcarbamylase deficiency, Osteogenesis imperfecta, Polycystic kidney disease, Rett syndrome, Retinoblastoma, Skeletal dysplasias, Smith Lemli Opitz, Spinal muscular atrophy, Spondylocostal dysostosis, TAR, Treacher Collins, Tuberous sclerosis 1/2, UPD 14, Wolcott-Rallison, X- linked Adrenoleukodystrophy and X-linked myotubular myopathy
Aneuploidy analysis: Various methods being developed and validated Quantitative analysis of SNPs in fetal specific mrna Extract RNA
Quantitative analysis of SNPs in fetal specific mrna PLAC 4 A / G A A G A Chr 21 Normal meiosis G A A A G A Non disjunction meiosis I (maternal) A G G A Normal A A A Trisomy 21
Quantitative analysis of SNPs in fetal specific mrna Analysis by MALDI-TOF (mass spectrometry) PLAC4 mrna ( ) is derived exclusively from fetal chromosome 21 PLAC4 mrna expressed in the placenta and is found in the plasma of pregnant women Normal fetus Normal fetus Trisomy 21 fetus Non informative 1 : 1 2 : 1 The Impact Lof et Genomics al., Nature on Public Med Health & PNAS, 25/04/2013 2007
Results of shotgun sequencing of maternal plasma DNA Trisomy 13 n=1 Trisomy 18 n=2 Trisomy 21 n=9 Fan et al., PNAS Oct 2008
ffdna: Patient and healthcare benefits Non-invasive testing Reduced risk of miscarriage Earlier testing availability Significantly reduced cost of PND Reduction in invasive procedures
Next Generation Sequencing (NGS) Ability to sequence whole genome or selected regions of the genome On a scale massively larger than Sanger sequencing Applications Whole genome (100kg) Whole exome Targeted (panels)
Sanger sequencing NGS
And time
Application Example(s) Benefits over existing technology Screening multiple genes (in multiple patients) to determine treatment, surveillance Cardiac, cancer. Rare, specialist genetic conditions Turnaround, cost, high throughput analysis, sensitivity Provision of UK (and European) services. Whole genome sequencing Rare unknown syndromes Avoids unnecessary dx tests, answers & risk assessment for families Translational R&D Clinical trials, commercial services High throughput, cost, broad molecular characterisation Enhancement of existing services Invasive prenatal testing for Down s replaced by maternal blood test Cost, acceptability
e.g. Cardiac genetics Benefits of identifying at-risk family members: Appropriate surveillance and treatment Reduced morbidity for those not at risk Significant cost-savings Analysis of 13 genes Sanger sequencing NGS Cost 6500 1200 TaT 24m 2m Acceptability No Yes
The problems of NGS Detection of unclassified variants Unexpected findings ELSI Data storage However, it is a fantastic development for diagnostic services!!!
NGS: Patient and healthcare benefits Increased sensitivity of genetic analysis for rare inherited disorders Opportunity for testing for really rare unknown genetic disorders Vastly reduced reporting times Reduced anxiety Reduction in tests ( ) before clinical diagnosis is made Potential to develop new diagnostic services
Personalised medicine Stratified medicine in cancer Pharmacogenetics Adverse affects Drug metabolism
The challenge: drugs don t work for most cancer patients Drug Response Rate, % 80 60 Analgesics Cancer 40 20 0 Source: Spears et al., Trends Mol Med, 2001; Lazarou et al., JAMA, 1998, 27
...and we are struggling to produce new drugs The value of the largest 9 pharmaceutical companies fell by $0.6tn 2001-2009 http://www.ncbi.nlm.nih.gov/pmc/articles/pmc2953249/
But what s better for patients? benefits of personalised medicine
NSCLC Ref: T Mok IPASS trial. JCO 2009 27 (suppl):408s (abstract 8006) Progression-free survival in EGFR selected patients treated with Gefitinib
Exon 21 L858R mutation case study Iressa- 6 weeks & 4 months
Biomarkers that predict response in solid tumours Biomarker Tumour Drug / biomarker approved HER2 Breast / gastric Yes EGFR NSCLC Yes KRAS mcrc Yes (liver mets) c-kit / PDGFRA GIST Yes 1p36 / 19q13, MGMT, IDH1/2 Multiforme glioblastoma In common use BRAF (c-kit, NRAS) BRAF Metastatic melanoma (mcrc) Yes ALK-EML4 NSCLC 2013 MSI mcrc 2012-13 BRCA1/2 Breast? PTEN Prostate? PI3KCA mcrc?
Validated biomarkers predicting treatment or prognosis Solid tumours where either no or significant proportion of patients have no predictive or prognostic biomarker Tumour Germline
Pharmacogenetics Drug efficacy Adverse affects Pharmacodynamics the biochemical and physiological effects of drugs and the mechanisms of their actions Pharmacokinetics Pharmacokinetics, the movement and change of drugs in the body over a period of time
Warfarin Pharmacodynamics Pharmacokinetics
Warfarin dosing depends upon genotype at both VKORC1 and CYP2C9 loci
Well known pharmacogenetic associations Taken from http://www.pharmgkb.org
Personalised Medicine: Benefits for patients and Healthcare Treatment is tailored to the individual Should therefore work No / reduced side-effects (For cancer therapies) Oral drugs can be taken at home Considerably improved survival Significant cost-saving on drugs and readmissions
This is a snap-shot.. Many more service developments across UK Regional Genetic centres We re good at sharing!