A recurrent copy number variation of the NEB triplicate region: only revealed by the targeted nemaline myopathy CGH array

Abstract

Recently, new large variants have been identified in the nebulin gene (NEB) causing nemaline myopathy (NM). NM constitutes a heterogeneous group of disorders among the congenital myopathies, and disease-causing variants in NEB are a main cause of the recessively inherited form of NM. NEB consists of 183 exons and it includes homologous sequences such as a 32-kb triplicate region (TRI), where eight exons are repeated three times (exons 82-89, 90-97, 98-105). In human, the normal copy number of NEB TRI is six (three copies in each allele). Recently, we described a custom NM-CGH microarray designed to detect copy number variations (CNVs) in the known NM genes. The array has now been updated to include all the currently known 10 NM genes. The NM-CGH array is superior in detecting CNVs, especially of the NEB TRI, that is not included in the exome capture kits. To date, we have studied 266 samples from 196 NM families using the NM-CGH microarray, and identified a novel recurrent NEB TRI variation in 13% (26/196) of the families and in 10% of the controls (6/60). An analysis of the breakpoints revealed adjacent repeat elements, which are known to predispose for rearrangements such as CNVs. The control CNV samples deviate only one copy from the normal six copies, whereas the NM samples include CNVs of up to four additional copies. Based on this study, NEB seems to tolerate deviations of one TRI copy, whereas addition of two or more copies might be pathogenic.

An Extended Targeted Copy Number Variation Detection Array Including 187 Genes for the Diagnostics of Neuromuscular Disorders

“…These regions are typically excluded from commercial CGH-array designs because of the difficulty their repetitive nature imposes on the analysis. In terms of the NEB TRI region, our array has been validated [16], and we are currently validating the analysis of the SD region of TTN .…”

“…The number of false positives and false negatives is higher, and the findings still require validation by other methods, such as aCGH. Repetitive regions may harbour CNVs in unconventional numbers [16], with the baseline deviating from the normal diploid copy number of two, and these can be difficult to determine using NGS-based methods and read counts only. In addition, the CNV breakpoints may be pinpointed with higher precision using a densely tiled aCGH design compared with sequence-based methods.…”

“…Our array allows for the detection of CNVs, not only in NMD genes in general, but also in the repetitive NEB TRI region [16], and the SD region of TTN . These regions are typically excluded from commercial CGH-array designs because of the difficulty their repetitive nature imposes on the analysis.…”

“…In NEB , this region holds a threefold repetition of an 8-exon block (exons 82–89, 90–97 98–105), referred to as the triplicate region (TRI). The NEB TRI region is known to harbour CNVs – gains and losses of one 8-exon block are considered benign, while gains of more than one 8-exon block are considered to be pathogenic [16]. In TTN , the SD constitutes a threefold repetition of 9 exons (exons 166–174, 175–183, and 184–192) within the PEVK segment of the gene, followed by fourth copies of the first two exons of the repeated block [15].…”

“…In TTN , the SD constitutes a threefold repetition of 9 exons (exons 166–174, 175–183, and 184–192) within the PEVK segment of the gene, followed by fourth copies of the first two exons of the repeated block [15]. CNVs in this type of region are all but rare, and may potentially be pathogenic [16]. …”