Genetic Testing

Genetic testing for diagnostic and prognostic purposes is ever expanding in haematology. If, like me, you are/were a clinical trainee you may never have had practical exposure to the laboratory techniques involved. The notes below are my efforts to understand the tests and terminology when reading reports or speaking to the haematopathologists. This recent 2022 BSH guideline was the basis for much of the NGS section.

DNA glossary

Nucleotide - distinct subunit of DNA (A, C, G and T)

Base pair - two nucleotides bonded between the two strands of DNA (A+T or C+G)

Kilobase (kb) - 1,000 base pairs

Gene - the piece of DNA required for the synthesis of a protein, made up of exons +/- introns

Exon - coding sections of DNA

Introns - non-coding sections of DNA

karyotyping

Cells are cultured to induce metaphase and images taken whilst in this state.

Allows for visual assessment of the number and appearance of the chromosomes. Typically, 20 metaphases are assessed to form a report.

Good for detecting chromosomal imbalances - deletion, monosomy, duplication, trisomy, translocation

Fluorescent In Situ Hybridisation (FISH)

Cells are cultured to induce metaphase and fluorescent markers added which bind to a pre-determined gene / gene locus.

Samples are then examined under fluorescent light. Typically, 20 metaphases are assessed to form a report.

Good to identify specific chromosomal abnormalities, some with short turnaround times. E.g. PML-RARA / t(15;17) to confirm diagnosis of APML

Can also be used to assess small ‘panels’ of recurrent abnormalities of prognostic significance, e.g. at diagnosis in myeloma.

Sanger Sequencing

Original method for determining the nucleotide sequence of DNA. Still in active use.

Requires a primer (single stranded DNA template) from which to start the ‘read’ of the DNA being tested

Next Generation (high throughput) Sequencing (NGS)

Reading NGS Reports

Variant genes will very often be identified in normal individuals, and the chance increases with the number of genes analysed.

Variants should therefore be reported with some clinical interpretations, common terms include:

  • Pathogenic variant

  • Likely pathogenic variant

  • Variant of uncertain significance (VUS)

  • Likely benign

  • Benign

Detected variants will usually be reported along with their variant allele frequency (VAF)

  • This is the % of reads that contain the variant of interest as a proportion of total reads

  • A VAF <5% is unlikely to be clinically significant

  • A VAF of 50% raises the possibility of an heterozygous germline variant. In practice, due to the nature of the PCR technology used, a VAF ‘approaching 50%’ (>30%) should be reviewed for consideration of germline origin.

Targeted Sequencing (t-NGS)

A selected panel of approx. 20-200 genes, e.g. the myeloid gene panel your local haematopathologists run for new AML/MDS diagnoses.

Method

  • Capture chosen exons

  • Amplification

  • Sequencing

Advantages

  • Cost

  • Relative ease of interpretation

  • Few unsolicited findings

Disadvantages

  • Only tests targeted regions —> mutations may be missed

  • Harder to identifiy copy number variants

Single Nucleotide Polymorphism (SNP) Array

A type of high throughput sequencing

Often used in combination with results of karyotyping, or as an alternative when karyotyping fails

Good for detecting chromosomal imbalances, e.g. deletions, duplications, unbalanced translocations

Whole Exome Sequencing (WES)

Exome = 30,000 exons of known coding genes (only a subset of these are analysed in the report)

Covers 1.5% of genome, but 80-90% of known pathogenic mutations

Method

  • Capture all exons

  • Amplification

  • Sequencing

Advantages

  • Cheaper than WGS

Disadvantages

  • High chance of unsolicited findings (and accompanying ethical issues)

  • May not detect Copy Number Variants (CNV)

Whole Genome Sequencing (WGS)

All genes and intergenic regions (only a subset of these are analysed in the report)

Method

  • DNA fragmented at random

  • Direct sequencing (i.e. without amplification)

Advantages

  • Entire genome tested

  • Better chance of detecting Copy Number Variants (CNV)

Disadvantages

  • Cost

  • High chance of unsolicited findings (and accompanying ethical issues)

Additional Glosary

Somatic mutation - a mutation found within the malignant cell of interest, which has been acquired during the patient’s lifetime

Germline mutation - a mutation found within non-cancerous cells of the body, which may indicate an hereditary cancer predisposition

Copy Number Variants (CNV) - large duplications and deletions involving whole gene(s)

In Frame Mutation - a mutation that means the DNA sequence can still be read and a protein produced. This protein will be abnormal but may still be partially functional.

Loss of Heterozygosity (LOH) - there should be two alleles at any particular gene locus. In a heterozygous state, one allele is normal and one is abnormal. If an acquired abnormality leads to the normal allele being lost then only one (abnormal) allele remains. This is called loss of heterozygosity.

Additional resources

2023 How I Treat paper on communication germline testing to patients. Several useful tables. (paywall, sadly)

Home - OMIM - Online Mendelian Inheritance in Man - genetic catalog

ClinVar (nih.gov) - genetic variants