Discrepancies in reporting the CAG repeat lengths for Huntington's disease

Abstract

on behalf of the European Huntington's Disease Network Huntington's disease results from a CAG repeat expansion within the Huntingtin gene; this is measured routinely in diagnostic laboratories. The European Huntington's Disease Network REGISTRY project centrally measures CAG repeat lengths on fresh samples; these were compared with the original results from 121 laboratories across 15 countries. We report on 1326 duplicate results; a discrepancy in reporting the upper allele occurred in 51% of cases, this reduced to 13.3% and 9.7% when we applied acceptable measurement errors proposed by the American College of Medical Genetics and the Draft European Best Practice Guidelines, respectively. Duplicate results were available for 1250 lower alleles; discrepancies occurred in 40% of cases. Clinically significant discrepancies occurred in 4.0% of cases with a potential unexplained misdiagnosis rate of 0.3%. There was considerable variation in the discrepancy rate among 10 of the countries participating in this study. Out of 1326 samples, 348 were re-analysed by an accredited diagnostic laboratory, based in Germany, with concordance rates of 93% and 94% for the upper and lower alleles, respectively. This became 100% if the acceptable measurement errors were applied. The central laboratory correctly reported allele sizes for six standard reference samples, blind to the known result. Our study differs from external quality assessment (EQA) schemes in that these are duplicate results obtained from a large sample of patients across the whole diagnostic range. We strongly recommend that laboratories state an error rate for their measurement on the report, participate in EQA schemes and use reference materials regularly to adjust their own internal standards.

Huntingtin gene CAG repeat numbers in Chinese patients with Huntington's disease and controls

“…Potential variations in CAG numbers between laboratories may be a limitation of such a multicentre survey, but this is a common finding, as demonstrated in a systematic comparative analysis in the European Huntington's Disease Network [37], and the similarity of our data confirms the general validity of our interpretation. The low number of intermediate and low penetrance CAG range does not allow firm conclusions about the phenotype in this group.…”

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

“…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. Studies have shown that in clinical HD analysis, 3%–13% of alleles fall outside error limits set by generally adapted best practice guidelines (Losekoot et al., ; Quarrell et al., ). An exact determination of the repeat count is important for clinical diagnostics of HD patients, in particular for the cases where repeat sizes cross borders of the repeat size ranges correlated with disease phenotypes.…”

“…Alleles up to 26 repeats are considered normal, while 27–35 repeats are intermediate alleles with potential to expand into the disease range in the next generation. Alleles with 36–39 CAG repeats are HD‐causing alleles with reduced penetrance, and the patient may or may not develop HD, and while alleles with ≥40 repeats are full‐penetrance HD‐causing alleles (Losekoot et al., ; Palomaki & Richards, ; Quarrell et al., ).…”

Phenotype Characterization of HD Intermediate Alleles in PREDICT-HD

“…Once we have more specific information regarding the factors that impact phenotype expression in the IA and RP ranges, it might become increasingly important to more accurately report the length of a person’s longest CAG repeat allele. Little attention is given in the literature to the issue of inconsistent reporting of CAG repeat lengths, which occurs in up to 51% of tests [33]. The ACMG/ASHG guidelines state that acceptable error rates for CAG repeat lengths is ± 2 repeats for alleles with less than 50 repeats [3].…”