Introduction
(i) Chromosomes in genetic diagnostics
Chromosomes visualized by classical and banding cytogenetics
Feature | GTG banding | FISH | CMA | NGS |
---|---|---|---|---|
Principle
| Monochrome-stained chromosomes are screened for numerical, structurally balanced and unbalanced aberrations | DNA-specific fluorescent probes are hybridized on patient chromosomes/nuclei to resolve structural and numerical aberrations | Patient DNA is applied to a microarray to screen for genomic gains or losses (and if SNP based for LOH) at high resolution | Panel-specific and/or genome-wide detection of causal mutations in monogenic disorders by high-throughput sequencing techniques |
Material
| Living cells | Metaphases and interphase | DNA | DNA |
Tissue sections, primary fixed cells | ||||
Equipment
| Incubator | FISH probes | Microarray | NGS platform |
Light microscope | Fluorescence microscope | Scanner | Bioinformatic data processing | |
Range
| Genome-wide | Genome-wide (M-FISH) | Genome-wide | Genome-wide (WGS) |
Single cell level | Locus- and region-specific | Pool of cells | Gene panel-specific | |
Single cell level | Pool of cells | |||
Resolution for routine diagnostics
| >5–10 Mb | >50 kb | >50 kb | 1–300 bp |
Mosaic detection (minimum range)
| Depends on the number of analyzed cells (5–25%) | Depends on the number of analyzed cells (0.1–1%) | Depends on the mosaic ratio in the sample (20–30%; SNP array <5%) | Depends on the mosaic ratio in the sample and coverage and the analysis software (>0.1% with MuTect; >10% with HaploTyper) |
Minimal turnaround time
| Depending on culture time: 1–15 days | Depending on FISH probe type: 3 h to 3 days | Depending on platform: 1–4 d | Depending on platform and panel: 5–14 d |
Applications
| First-line screening test for gross structural and numerical aberrations | Second-line test for specific rearrangements | Second-line screening for genomic imbalances after normal GTG/FISH | Second-/third-line panel-specific or genome-wide screening for DNA mutations |
Tumor cytogenetic | Detects small mosaics | Defining size of chromosomal imbalances | First-line test as panel in defined clinical subgroups | |
(Metaphases for FISH) | Verification of GTG and CMA results | Verification in parents to interpret VUS in offspring | Tumor-specific targeted panels | |
Resolves complex rearrangements | Predictive testing and verification of familial variants | |||
Parental screening for predisposing rearrangements | (CNV detection) | |||
Tumor-specific FISH panels | ||||
Advantage
| Low costs for equipment and consumables | Widely used for commercial probes | Widely used | High throughput lowers single-run/-case costs |
Widely used | Sensitive mosaic detection | ~100 × higher resolution than GTG | Widely used | |
Detection of heterochromatic regions | Predisposing rearrangements in parents detectable, genomic location and orientation resolvable | CNV detection | Single base resolution on genome-wide scale | |
Global whole genome view with information on genomic location | CNV detection (especially deletion, inversion, translocation events) at higher resolution than GTG | Low-level mosaics detectable | ||
Verification method | ||||
Use of frozen or FFPET sections | ||||
Pitfalls and disadvantages
| Viable cells and culture artefacts | Possible cross hybridization | Heterochromatic regions are not covered | Short reads limit CNV and structural aberration detection |
Small and/or rare rearrangements can be overlooked | No commercial FISH probe for region of interest | Location of an imbalance within the genome is unknown | Heterochromatic/repetitive regions are unsatisfactorily/not covered (artefact variants) | |
Experienced staff is needed | High costs of FISH probes | Balanced aberrations are not detectable | Fidelity at low coverage | |
Resolution | Small duplication events are hard to verify | Low-level mosaics escape | High rate of VUS, interpretation problems, secondary findings | |
FISH assay and lab-specific cut-offs need to be established | High rate of VUS | No standards for software pipelines and data processing | ||
Costs for equipment and consumables | Costs for equipment and consumables | |||
Polyploidy detection | Bioinformatic trained stuff is needed |