Molecular (cyto-) genetics Femke de Vries Clinical Laboratory - - PowerPoint PPT Presentation

Molecular (cyto-) genetics

Femke de Vries Clinical Laboratory Geneticist

Find genetic variation responsible for a specific disease in a patient

Aim

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
• Choose 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

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
• Choose 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

phenotypical description, family history

Kaiser et al. Hum Mol Genet. 2014 Jun 1; 23(11): 2888–2900.

Sarah B. Pierce et al. PNAS 2011;108:18313-18317

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

Genome- wide screen Confirmation experiment Targeted approach Confirmation experiment

Targeted vs Genome-wide

Whole genome analysis; resolution!

22q11

karyotyping

Hybridization

scanner

Arrays WGS

Chromosome level: karyotyping

DNA segment level: CNV-profiling

Nucleotide sequence level: WGS

Targeted analysis; previous knowledge

Fluorescent in situ hybridisatie (FISH)

Probe 21 22q11

MLPA

+2q

• 15q

Gene panels or WES Sanger sequencing qPCR or MAQ-assay

What do I want to detect?

Adapted from Speicher & Carter: The new cytogenetics: blurring the boundaries with molecular biology

• A. de Klein NIHES 2013

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

Karyotyping

Numerical, translocations, inversions, duplications and deletions (> 5Mb)

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, 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

Copy Number Loss /homozygous loss Copy Number Gain

Garland Science chapter 4: principles of genetic variation.

Most CNVs do not have clinical significance!

Santhosh Girirajan et al.; Human Copy Number Variation and Complex Genetic Disease

CGH-array and SNP-array

CNV and allelic information CNV information

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

Log2 ra rat io BAF AF

Illumina 610Q array/Genome studio software

B A BBB BBA BAA AAA 100% B BB ~ 50% B AB 0% B AA

Submicroscopic deletions/duplications

David Miller et al. Consensus Statement: Chromosomal Microarray Is a First-Tier Clinical Diagnostic Test for Individuals with Developmental Disabilities or Congenital Anomalies

• Technical

interpretation

• Validate with

second technique

True CNV

• In-house control

cohort

• Public databases
• Literature

Causality

• In unaffected

parents

• In affected

individuals

Inheritance

CNV-profiling

Technical artefacts QC Population frequency

WGS or WES analysis

Variants filtered if present more than X times in in-house cohort Keep variants only if quality parameters are moderate or good Filter if present in more than 3%-0.1%

• f chromosomes (public control databases)

• In-house control

cohort

• Public databases

Unaffected controls

• In unaffected

parents

• In affected

individuals

Inheritance

• Functional

consequence of variant

• Relationship with

diseasecandidate

Candidate variants

Exome-seq analysis

Preliminary analysis: Gene panel

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

FISH

• A. de Klein NIHES 2013

Classic cytogenetic techniques

Chromosome banding FISH (fluorescent insitu hybridisation) With FISH:

• prior knowledge essential
• small probes (40-200 kb)

Di-George syndrome

Deletion 22q11

22 22 FISH to detect known 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

3q gain no gain no gain ? ?

FISH index

FISH mother

Mechanism of inheritance

Mechanism of inheritance

Balanced carrier meiosis

N dup del bal ins

Family tree

3q gain normal balanced insertion 3q 3q gain 3q gain normal normal

De novo Copy Number Loss

Multiplex Amplicon Quantification

5 amplicons in region of interest, 6 amplicons in other genomic locations Multiplex: separation on amplicon length. CN state based on fluorescent intensity; normalisation on 4 controls

de novo C/T

Father Mother Patient

WGS/WES validate variants with Sanger-seq

CNV can unmask a gene mutation

Homozygous c.854 G>T in SCARF1 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

• 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
• Use a second –targeted- technique to validate your results

Take home messages