Molecular (cyto-) genetics Find genetic variation responsible for a - PowerPoint PPT Presentation

Aim Molecular (cyto-) genetics Find genetic variation responsible for a specific disease in a patient Femke de Vries Clinical Laboratory Geneticist What do I need? What do I need? • Clear phenotypical description, family history • Clear phenotypical description, family history • Blood, saliva, (or tissue of interest), DNA of patient and family • Blood, saliva, (or tissue of interest), DNA of patient and family • The proper technique to detect the expected variation • The proper technique to detect the expected variation • Compare your findings with unaffected controls • Compare your findings with unaffected controls • Choose the proper technique to confirm your initial findings • Choose the proper technique to confirm your initial findings • Determine the effect of the variation on RNA or protein • Determine the effect of the variation on RNA or protein • Link the disturbed protein function or expression to the disease • Link the disturbed protein function or expression to the disease

phenotypical description, family history What do I need? • Clear phenotypical description, family history • Blood, saliva, (or tissue of interest), DNA of patient and family • The proper technique to detect the expected variation • Compare your findings with unaffected controls • The proper technique to confirm your initial findings • Determine the effect of the variation on RNA or protein • Link the disturbed protein function or expression to the disease Sarah B. Pierce et al. PNAS 2011;108:18313-18317 Kaiser et al. Hum Mol Genet. 2014 Jun 1; 23(11): 2888–2900. Targeted vs Genome-wide Whole genome analysis; resolution! karyotyping WGS Genome- Targeted 22q11 wide screen approach Arrays Confirmation Confirmation experiment experiment Hybridization scanner

Chromosome level: karyotyping DNA segment level: CNV-profiling Nucleotide sequence level: WGS Targeted analysis; previous knowledge Gene panels or WES Sanger sequencing Fluorescent in situ hybridisatie (FISH) Probe 21 Probe be be 21 be be be be be be be 22q11 MLPA qPCR or MAQ-assay +2q -15q

A. de Klein NIHES 2013 What do I want to detect? Types of genomic variation Structural rearrangements, inversions, duplications and deletions At least 5 Mb in length Segmental duplications & Copy Number Variation 50 bp in length, usually more than 1kb Microsatellite, minisatellite & satellite DNA Tandemly repeated sequences, repetitive DNA 100bp to hundreds of kb Small Insertions and Deletions (InDel) Up to ~50 bp in length Single Nucleotide Variants (SNV) Substitution of single nucleotides Adapted from Speicher & Carter: The new cytogenetics: blurring the boundaries with molecular biology Karyotyping Types of genomic variation Numerical, translocations, inversions, duplications and deletions (> 5Mb) Structural rearrangements, inversions, duplications and deletions At least 5 Mb in length Segmental duplications & Copy Number Variation 50 bp in length, usually more than 1kb Microsatellite, minisatelite & satelite DNA Tandemly repeated sequences, repetitive DNA 100bp to hundreds of kb Small Insertions and Deletions (InDel) Up to ~50 bp in length Single Nucleotide Variants (SNV) Substitution of single nucleotides

Copy Number Variations CGH-array and SNP-array Copy Number Loss /homozygous loss Copy Number Gain Garland Science chapter 4: principles of genetic variation . Most CNVs do not have clinical significance ! CNV information CNV and allelic information Santhosh Girirajan et al.; Human Copy Number Variation and Complex Genetic Disease Addapted from: E. Karampetsou et al.; Microarray Technology for the Diagnosis of Fetal Chromosomal Aberrations: Which Platform Should We Use? Arrays : SNP arrays: B-allele frequency BAF Submicroscopic deletions/duplications Illumina 610Q array/Genome studio software Log2 rat io BBB B 100% B BB BBA BAF ~ 50% B AB BAA AAA A 0% B AA David Miller et al. Consensus Statement: Chromosomal Microarray Is a First-Tier Clinical Diagnostic Test for Individuals with Developmental Disabilities or Congenital Anomalies

WGS or WES analysis CNV-profiling Technical Variants filtered if present more than artefacts X times in in-house cohort Causality • Technical • In unaffected interpretation parents • In-house control Keep variants only if quality • Validate with • In affected parameters are moderate or good cohort QC second technique individuals • Public databases • Literature True CNV Inheritance Filter if present in more than 3%-0.1% Population of chromosomes (public control databases) frequency Preliminary analysis: Gene panel Exome-seq analysis Inheritance • Functional consequence of • In-house control • In unaffected variant cohort parents • Relationship with • Public databases • In affected diseasecandidate individuals Unaffected Candidate controls variants

A. de Klein NIHES 2013 FISH What do I need? • Clear phenotypical description, family history Di-George syndrome Classic cytogenetic techniques • Blood, saliva, (or tissue of interest), DNA of patient and family Chromosome banding 22 FISH (fluorescent insitu hybridisation) • The proper technique to detect the expected variation 22 • Compare your findings with unaffected controls With FISH: - prior knowledge essential - small probes (40-200 kb) • The proper technique to confirm your initial findings Deletion 22q11 • Determine the effect of the variation on RNA or protein FISH to detect known • Link the disturbed protein function or expression to the disease disease related deletions Confirmation of variation Copy number gain Patient with intellectual disability and minor congenital anomalies Targeted array parents: no gain � “de novo”

Family tree FISH index no gain no gain ? ? 3q gain FISH mother Mechanism of inheritance meiosis Mechanism of inheritance Balanced carrier N dup del bal ins

Family tree De novo Copy Number Loss balanced normal 3q gain 3q gain insertion 3q 3q gain normal normal Multiplex Amplicon Quantification WGS/WES validate variants with Sanger-seq Father Mother Patient 5 amplicons in region of interest, 6 amplicons in other genomic locations de novo C/T Multiplex: separation on amplicon length. CN state based on fluorescent intensity; normalisation on 4 controls

CNV can unmask a gene mutation Take home messages � Single basepair changes are not the only genomic variation � Our genomes are highly variable; even large deletions or duplications can occur; these can be disease causing or can also be harmless � There are many techniques; choose the one best fitting your question � Use a genome wide technique to detect unknown variation Homozygous c.854 G>T in SCARF1 � Use a second –targeted- technique to validate your results Recessive disorder; van den Ende-Gupta syndrome Bedeschi et al.; Unmasking of a Recessive SCARF2 Mutation by a 22q11.12 de novo Deletion in a Patient with Van den Ende-Gupta Syndrome