Tools for human molecular diagnosis Joris Vermeesch
Chromosome > DNA
Genetic Code
Effect of point mutations/polymorphisms
Effect of deletions/insertions
Effect of splicing mutations IVS2-2A>G Normal splice site Exon 1 Exon 2...CCCCACATAgtaagg......gagtagCCATTCCACAGATTTT......CCCCACATAgtaagg... Cryptic splice site...gagtggccattccacagatttt...
Genetic testing 1 gene <> multiple genes Molecular testing 1 disease 1 (single) mutation 1 disease 1 gene different diseases 1 (single) gene (un)known defect karyotyping 1 disease few genes 1 disease 1 chromosomal anomaly different diseases many genes different diseases different anomalies Molecular cytogenetic testing total genome Cytogenetics
How do we test which tools to use? Mutation-specific test - Restriction enzyme digestion - Southern blot - Dot blot / reverse dot blot - PCR hybridization - PCR applications PCR/fragment sizing MLPA: large deletion/duplications Mutation detection - Mutation scanning o SSCP o DGGE o DHPLC o HRM-CA - Sequencing o Sanger sequencing o Next Generation Sequencing
How do we test which tools to use? Mutation-specific test - Restriction enzyme digestion - Southern blot - Dot blot / reverse dot blot - PCR hybridization - PCR applications PCR/fragment sizing MLPA: large deletion/duplications Mutation detection - Mutation scanning o SSCP o DGGE o DHPLC o HRM-CA - Sequencing o Sanger sequencing o Next Generation Sequencing
Factor V (Leiden mutatie 1691A) Mnl I 5 - GGCGAGG 3 5 - GGCAAGG 3 Arg(R) to Gln(Q) at position 506-267 -200-163 -67-37 37 163 67 200 67
How do we test which tools to use? Mutation-specific test - Restriction enzyme digestion - Southern blot - Dot blot / reverse dot blot - PCR hybridization - PCR applications PCR/fragment sizing MLPA: large deletion/duplications Mutation detection - Mutation scanning o SSCP o DGGE o DHPLC o HRM-CA - Sequencing o Sanger sequencing o Next Generation Sequencing
Southern blot
Southern blot FMR
How do we test which tools to use? Mutation-specific test - Restriction enzyme digestion - Southern blot - Dot blot / reverse dot blot - PCR hybridization - PCR applications PCR/fragment sizing MLPA: large deletion/duplications Mutation detection - Mutation scanning o SSCP o DGGE o DHPLC o HRM-CA - Sequencing o Sanger sequencing o Next Generation Sequencing
Distribution of Cystic Fibrosis Mutations 1 2 3 4 5 6a 6b 7 8 9 10 11 12 13 14a 14b 15 16 17a 17b 18 19 20 21 22 23 24 5' 3' Missense AA deletion Nonsense Frameshift Splice site Amino acid Variation Total Membrane spanning ATP-binding R-domain Membrane spanning ATP-binding (Data from the CF Genetic Analysis Consortium- July, 1995)
F508del (ex.10) 72.5 % G542X (ex.11) 5.5 % N1303K (ex.21) 3.5 % 1717-1G>A (in. 10) 2.5 % W1282X (ex.20) 1.0 % G551D (ex.10) 0.5 % R553X (ex.10) 0.5 % I 507del (ex.10) 0.5 %
Reverse Dot Blot principe chromogeen paars precipitaat alkalisch fosfatase streptavidine biotine geamplificeerd DNA DNA-probe membraan
How do we test which tools to use? Mutation-specific test - Restriction enzyme digestion - Southern blot - Dot blot / reverse dot blot - PCR hybridization - PCR applications PCR/fragment sizing MLPA: large deletion/duplications Mutation detection - Mutation scanning o SSCP o DGGE o DHPLC o HRM-CA - Sequencing o Sanger sequencing o Next Generation Sequencing
TaqMan
TaqMan output
Molecular beacon
FRET (Fluorescence Resonance Energy Transfer) Principe
FRET (Fluorescence Resonance Energy Transfer) Principle
FRET (Fluorescence Resonance Energy Transfer) Principle
How do we test which tools to use? Mutation-specific test - Restriction enzyme digestion - Southern blot - Dot blot / reverse dot blot - PCR hybridization - PCR applications PCR/fragment sizing MLPA: large deletion/duplications Mutation detection - Mutation scanning o SSCP o DGGE o DHPLC o HRM-CA - Sequencing o Sanger sequencing o Next Generation Sequencing
MLPA: Multiplex Ligation-dependent Probe Amplification
1,2 1 0,8 0,6 0,4 0,2 0 MLH1 exon 1 MSH2 exon 1 MLH1 exon 2 MSH2 exon 2 MLH1 exon 3 MSH2 exon 3 MLH1 exon 4 MSH2 exon 4 MLH1 exon 5 MSH2 exon 5 MLH1 exon 6 MSH2 exon 6 MLH1 exon 7 MSH2 exon 7 MLH1 exon 8 MSH2 exon 8 MLH1 exon 9 MSH2 exon 9 MLH1 exon 10 MSH2 exon 10 MLH1 exon 11 MSH2 exon 11 MLH1 exon 12 MSH2 exon 12 MLH1 exon 13 MSH2 exon 13 MLH1 exon 14 MSH2 exon 14 MLH1 exon 15 MSH2 exon 15 MLH1 exon 16 MSH2 exon 16 MLH1 exon 17 MLH1 exon 18 MLH1 exon 19 MLPA MSL1&2
MLPA - SMA normal patient - SMA
How do we test which tools to use? Mutation-specific test - Restriction enzyme digestion - Southern blot - Dot blot / reverse dot blot - PCR hybridization - PCR applications PCR/fragment sizing MLPA: large deletion/duplications Mutation detection - Mutation scanning o SSCP o DGGE o DHPLC o HRM-CA - Sequencing o Sanger sequencing o Next Generation Sequencing
SSCP: Single strand conformation polymorphism
DGGE: Denaturing gradient gel electrophoresis Denaturant (Formamide/Urea) 0% 100% Separation Based on Differences in Nucleotide Sequence (G+C content) and Melting Characteristics Partially melted Single strands Or with GC-clamp Electrophoresis Double strand
DHPLC: Denaturing high performance liquid chromatography wild type mutant heteroduplexes homoduplexes A T G C A C G T A T G C
HRM: High resolution melting curve analysis wild type mutant heteroduplexes homoduplexes A T G C A C G T A T G C
How do we test which tools to use? Mutation-specific test - Restriction enzyme digestion - Southern blot - Dot blot / reverse dot blot - PCR hybridization - PCR applications PCR/fragment sizing MLPA: large deletion/duplications Mutation detection - Mutation scanning o SSCP o DGGE o DHPLC o HRM-CA - Sequencing o Sanger sequencing o Next Generation Sequencing
Sanger sequencing
Sequencing - ddntp
Sanger sequencing universal primers e1f 5 UTR e2f e3af e3bf e4/5f e6f Exon 1 Exon 2 Exon 3 Exons 4/5Exon 6 500bp 3 UTR 18bp M13-F e1r e2r e3ar e3br e4/5r e6r A30V intron 1 V L A Q D Y Q Q intron 2 ctagctattacgctactagc ctttccccag. gtc CTG GCT CAG gac tac cag cag.gtgggtatgc ctagctattacgctactagc 198C>T 18bp M13-R
Asp 90 G C T G A C A A A Asp/Ala 90 G C T G C C A A A A Controle Patiënt
Hereditarybreastand ovariancancer BRCA1 /BRCA2 BRCA1(on chromosome 17) BRCA2(on chromosome 13) 5.6 kb, 1863 aa - Affected patients identify the disease-causing mutation in the family - Dominantly inherited heterozygous mutations - Scattered thoughout the genes whole coding regions analyzed - Mostly truncating(nonsense, frameshifts(indels)) 10.3 kb, 3418 aa
Apostolou P, 2013
100 families
Aangepast van Fackenthal JD and Olopade OI, Nature Rev Cancer (2007) 185delAG IVS5+3A>G c.2197del5 c.2359dup c.3481del11 c.5266dupc IVS6+1G>A c.5213del4 c.6270del2 c.6275del2 c.8243g> A Exon19d el c.8904del
Vroeger Per patiënt= 110 PCR stalen 220 sequencing reacties Elke dag= 5 PCR platen 10 sequencing platen
BRCA MASTR v2.0 (Titanium) : 94 amplicons 20-50 ng per multiplex PCR reaction
ROI Extra Specifieke primer Universeel deel Index 1 Complementair aan seq primer
Genetictesting Molecular testing 1 disease 1 (single) mutation 1 disease 1 gene different diseases 1 (single) gene (un)known defect karyotyping Cytogenetics 1 disease few genes 1 disease 1 chromosomal anomaly different diseases many genes different diseases different anomalies Molecular cytogenetic testing total genome
Projecten in het laboratorium Massive Parallel Sequencing (Illumina HiSeq 2000-2500) Read Alignment, Mapping, Variant Calling and Annotation (Annovar) Variant Filtering (trio analysis) + CNV database mining IlluminaLibrary Prep and Exome Enrichment (NimbleGen) DE NOVO VARIANTS IN KNOWN MC GENES (8 PTS) DIAGNOSTIC VALUE e.g. CASK, CREBBP, TUBG1, KIF11 SYNDROMIC SPORADIC MC COHORT (19/20 trios) DE NOVO VARIANTS IN CANDIDATE MC GENES (7 PTS) e.g. LMNB1, SEMA4B, PKDL1, SNX21, SBSPON, FAT4, CCDC51 DE NOVO MUTATIONS TESTING IN ZEBRAFISH NO CANDIDATE DE NOVO VARIANTS (3 PTS)
Identification of causative mutations in rare disorders
Diagnostische exoom sequencing 100 patients 79 de novo mutations Diagnostic yield: 16% 51patients 87 de novo mutations Diagnostic yield: 45-55%
Limitations?! Exome is not entirely covered Limited to exons (and flanking regions) Impossible to detect (deep) intronic mutations Not quantitative Impossible to detect deletions or duplications Thousands of SNPs and variants
Gen en variant prioritisatie om causale SNPs te detecteren. Sifrim et al., Nature Methods, 2013
Volledige genoom analyse
Volledige genoom analyse 1.58 exomic variants/individual/generation Diagnostic yield: 62%
Wat met genetica in de maatschappij?