EMQN/CMGS best practice guidelines for the molecular genetic testing of Huntington disease

Abstract

Huntington disease (HD) is caused by the expansion of an unstable polymorphic trinucleotide (CAG)n repeat in exon 1 of the HTT gene, which translates into an extended polyglutamine tract in the protein. Laboratory diagnosis of HD involves estimation of the number of CAG repeats. Molecular genetic testing for HD is offered in a wide range of laboratories both within and outside the European community. In order to measure the quality and raise the standard of molecular genetic testing in these laboratories, the European Molecular Genetics Quality Network has organized a yearly external quality assessment (EQA) scheme for molecular genetic testing of HD for over 10 years. EQA compares a laboratory's output with a fixed standard both for genotyping and reporting of the results to the referring physicians. In general, the standard of genotyping is very high but the clarity of interpretation and reporting of the test result varies more widely. This emphasizes the need for best practice guidelines for this disorder. We have therefore developed these best practice guidelines for genetic testing for HD to assist in testing and reporting of results. The analytical methods and the potential pitfalls of molecular genetic testing are highlighted and the implications of the different test outcomes for the consultand and his or her family members are discussed.

American College of Medical Genetics and Genomics Standards and Guidelines for Clinical Genetics Laboratories, 2014 edition: technical standards and guidelines for Huntington disease

“…Normal alleles are defined as alleles with ≤26 CAG repeats. [5][6][7] These alleles are not pathologic and segregate as a stable polymorphic repeat in >99% of meiosis (Figure 1). The most common normal allele lengths contain 17 and 19 CAG repeats.…”

“…The most common normal allele lengths contain 17 and 19 CAG repeats. 5 Mutable normal alleles. Mutable normal alleles are defined as alleles with 27-35 CAG repeats, and this repeat range is often referred to as the meiotic instability range, or "intermediate alleles. "…”

Detailed analysis of HTT repeat elements in human blood using targeted amplification-free long-read sequencing

“…HD, as well as other trinucleotide repeat disorders, is typically diagnosed using PCR amplification of the repeat element, and the fragment size is determined by capillary electrophoresis. For very large expansions, Southern blotting protocols or triplet repeat primed PCR (TP‐PCR) are recommended as complementary technologies (Losekoot et al., ). Fragment analysis, as well as Southern blotting and TP‐PCR, is dependent upon accurate amplification and fragment sizing and does not analyze the DNA sequence itself.…”

Monitoring Huntington's Disease Mortality across a 30-Year Period: Geographic and Temporal Patterns

“…Diagnosis and genetic confirmation improved with the mapping of HD gene chromosome 4p in 1983 and the identification of the pathogenic mutation ten years later [4][5][6] . The current gold standard for the diagnosis is the DNA determination, showing a CAG-repeat of at least 36 on the huntingtin gene on chromosome 4p according to international guidelines [7,8] . The fact that HD has an estimated worldwide prevalence of around 2.7 cases per 100,000 persons (95% CI 1.55-4.72), means that it meets the criteria for being classified as a rare disease (RD) [9] .…”