Short tandem repeat (STR) testing

Short tandem repeats (STRs) occur when a short sequence of DNA is repeated many times in a row – for example, a triplet repeat such as CAG. These occur throughout the genome, often with little or no consequence. Some occur in functional regions, however, and if there are an unusual number of repeats (usually too many) this can have functional consequences.

STR testing is used to diagnose conditions that are predominantly caused by expansions (increases) in the length of a specific repetitive region of the genome. This includes well-known conditions such as Huntington disease (caused by a CAG trinucleotide repeat expansion in the HTT gene) and fragile X syndrome (caused by a CGG trinucleotide repeat expansion in the 5’ UTR of the FMR1 gene). Carrier testing and predictive testing for such conditions is also done by STR testing.

STR testing may also be used alongside next-generation sequencing (NGS) for patients with neurological symptoms for which the genetic cause is unclear. This is because, although NGS is useful to test many genes for many types of variant at the same time, it cannot yet size all STRs accurately. Improvements in sizing STRs from whole genome sequencing (WGS) data, and the advent of long-read sequencing technologies, may improve this in the near future.

There are two commonly used methods for sizing repeat expansions. These are outlined below.

• Fluorescent polymerase chain reaction (PCR) sizing (also known as fragment analysis).
  • Patient DNA is amplified using PCR.
  • The primers are located either side of the repeat region, so that the length of the PCR product reflects the length of the repeat.
  • One of the primers is fluorescently labelled.
  • Capillary electrophoresis is used to separate PCR products by size and fluorescence of the PCR product is detected.
• Repeat-primed PCR (can amplify larger expansions).
  • Patient DNA is amplified using PCR.
  • One primer is located next to the repeat region and is fluorescently labelled.
  • A second primer targets the repeat itself. This binds at multiple locations in the repeat region, generating multiple products, with the largest products reflecting the size of the entire expansion. This primer is present in limited amounts, so that a third primer (more abundant) takes over after a few PCR cycles.
  • This third primer targets the PCR products generated by the previous two, amplifying all of them.
  • Capillary electrophoresis is used to separate PCR products by size and fluorescence of the PCR products is detected.

Advantages

• It is a gold standard method for accurate sizing of repeat expansions.
• It is cost effective.
• It is less laborious and has higher throughput than Southern blotting.
• It may detect mosaicism for different repeat sizes (repeat-primed PCR).
• It may detect interruptions of the repeat expansion (repeat-primed PCR), for example where there is an AGG somewhere within a region of CGG repeats. There is evidence that these interruptions affect how likely a repeat is to expand in the next generation. Long-read sequencing may enable even more accurate detection of these interruptions in future.

Limitations

• For large expansions, exact sizing may not be possible.
• It cannot detect methylation status.
• It provides targeted testing for one repeat at a time. Improvements in sizing some STRs from WGS data mean that WGS can now be used for sizing multiple STRs at the same time, alongside the other variant types detected by WGS.
• False negatives can be caused by single nucleotide variants in a primer binding site (labs mitigate this risk by using familial positive controls, performing fluorescent PCR sizing with two different primer sets, or performing repeat-primed PCR in both directions).

• The samples required depend on the reason for testing.
  • A blood sample in an EDTA tube is required. From adults, 5–10ml of blood is required; 2–5ml is required from children; and from babies, 1–2ml is required.
  • Prenatal testing must be arranged in advance through a clinical genetics department. Chorionic villus samples in transport medium in a sterile leak-proof container or amniotic fluid in a sterile leak-proof universal container should be sent to cytogenetics for dissecting and culturing, with instructions to forward the sample to the regional molecular genetics laboratory for analysis.
• Target reporting time from receipt of sample is from 14 days to 42 days, depending on the clinical reason for the test.

• STRs occur when a short sequence of DNA is repeated multiple times in a row – for example, a triplet repeat such as CAG.
• STR testing is used to diagnose conditions that are predominantly caused by expansions (increases) in the length of a specific repetitive region of the genome.
• STR testing is a cost effective gold-standard method for accurate sizing of repeat expansions.
• STR testing can be carried out using fluorescent PCR or repeat-primed PCR.

For clinicians

• Asuragen: Example of a commercial repeat-primed PCR assay (AmplideX)
• National Human Genome Research Institute: Tandem repeat
• University of Toronto: Repeat expansion diagnostic techniques part 2 – repeat primed PCR (video, 9 minutes 20 seconds)

References:

• Fan H and Chu J-Y. ‘A brief review of short tandem repeat mutation‘. Genomics, Proteomics & Bioinformatics 2007: volume 5, issue 1, pages 7–14. DOI: 10.1016/S1672-0229(07)60009-6