Fine-tiling array CGH to improve diagnostics for α- and β-thalassemia rearrangements

Abstract

Implementation of multiplex ligation-dependent probe amplification (MLPA) for thalassemia causing deletions has lead to the detection of new rearrangements. Knowledge of the exact breakpoint sequences should give more insight into the molecular mechanisms underlying these rearrangements, and would facilitate the design of gap-PCRs. We have designed a custom fine-tiling array with oligonucleotides covering the complete globin gene clusters. We hybridized 27 DNA samples containing newly identified deletions and nine positive controls. We designed specific primers to amplify relatively short fragments containing the breakpoint sequence and analyzed these by direct sequencing. Results from nine positive controls showed that array comparative genomic hybridization (aCGH) is suitable to detect small and large rearrangements. We were able to locate all breakpoints to a region of approximately 2 kb. We designed breakpoint primers for 22 cases and amplification was successful in 19 cases. For 12 of these, the exact locations of the breakpoints were determined. Seven of these deletions have not been reported before. aCGH is a valuable tool for high-resolution breakpoint characterization. The combination of MLPA and aCGH has lead to relatively cheap and easy to perform PCR assays, which might be of use for laboratories as an alternative for MLPA in populations where only a limited number of specific deletions occur with high frequency.

Disease-targeted sequencing: a cornerstone in the clinic

“…For example, testing using long-range PCR can be carried out in parallel to compensate for inadequacy in NGS of genes with pseudogenes, such as STRC 14 . In addition, laboratories often supplement disease-targeted sequencing tests with copy number detection approaches to detect heterozygous and homozygous large deletions and other copy number changes, a category of mutations that is currently missed by exome-sequencing services 15 . This example highlights the basis for recent recommendations from the American College of Medical Genetics and Genomics that suggest that exome or genome sequencing approaches should be reserved for those cases in which disease-targeted testing is negative or unlikely to return a positive result in a timely and cost-effective manner 16 .…”

Thalassemia Intermedia Caused by 16p13.3 Sectional Duplication in a β-Thalassemia Heterozygous Child

“…MLPA or array CGH, or the combination of MLPA and CGH, is a reliable method to screen for α and β gene rearrangements in thalassemia and hemoglobinopathy [18][19][20]. In this study, we first screened for the α gene copy number alterations using MLPA.…”

Detection of Anti-Lepore Hb P-Nilotic by Multiplex Ligation-Dependent Probe Amplification

“…Because the distances between the probes used generally range between 3 and 10 kb, additional long-range PCR and sequencing are required to define the breakpoints of the deletion. Until more sophisticated techniques such as CGH array become more widely available, MLPA remains an efficient and sensitive adjunct to molecular testing for rare or unknown Hb variants resulting from globin gene rearrangements (9).…”