Expansion of a 12-kb VNTR containing the REXO1L1 gene cluster underlies the microscopically visible euchromatic variant of 8q21.2

Abstract

Copy number variants visible with the light microscope have been described as euchromatic variants (EVs) and EVs with extra G-light material at 8q21.2 have been reported only once before. We report four further patients with EVs of 8q21.2 ascertained for clinical (3) or reproductive reasons (1). Enhanced signal strength from two overlapping bacterial artificial chromosomes (BACs) and microarray analysis mapped the EV to a 284-kb interval in the reference genome. This interval consists of a sequence gap flanked by segmental duplications that contain the 12-kb components of one of the largest Variable Number Tandem Repeat arrays in the human genome. Using digital NanoString technology with a custom probe for the RNA exonuclease 1 homologue (S. cerevisiae)-like 1 (REXO1L1) gene within each 12-kb repeat, significantly enhanced diploid copy numbers of 270 and 265 were found in an EV family and a median diploid copy number of 166 copies in 216 controls. These 8q21.2 EVs are not thought to have clinical consequences as the phenotypes of the probands were inconsistent, those referred for reproductive reasons were otherwise phenotypically normal and the REXO1L1 gene has no known disease association. This EV was found in 4/3078 (1 in 770) consecutive referrals for chromosome analysis and needs to be distinguished from pathogenic imbalances of medial 8q. The REXO1L1 gene product is a marker of hepatitis C virus (HCV) infection and a possible association between REXO1L1 copy number and susceptibility to HCV infection, progression or response to treatment has not yet been excluded.

Figure 1

Idiogram of a normal chromosome 8 and partial G-banded karyotypes of the EV chromosomes 8 with the variant chromosome on the right in each case and the additional G-light material indicated by the black arrow: (a) Patient 1; (b) Patient 2; (c) Patient 3; (d) Patient 4 and (e) the father of Patient 4.

Figure 2

(a–e) FISH and microarray results: (a, b) single-colour FISH with BACs RP11-90G23 (green) and BAC RP11-96G1 (red) in metaphases and an interphase nucleus from Patient 1 with the variant chromosome indicated by the large arrows; (c) dual-colour FISH with BACs RP11-90G23 (green) and RP11-96G1 (red) in an interphase nucleus from Patient 1 showing the colocalisation of the combined signals (yellow); (d) FISH with BAC RP11-96G1 (red) in a prometaphase from Patient 1 showing the lack of separation of the signals on the variant chromosome; (e) idiogram of chromosome 8 showing the location of the CNV region in distal 8q21.2; (f) Affymetrix Cytoscan HD array analysis of base pairs 86 460 508–87 259 456 in Patient 4 using the Affymetrix ChAS Software. The normal copy number results cluster along the horizontal weighted log₂ ratio=0 line and flank the variant 284 kb region between base pairs 86 553 129 and 86 836 909 (hg19). The horizontal lines above and below represent log₂ ratios of +1 and −1, respectively. (g) A Portable Document Format (PDF) screen shot of the variant and flanking regions of 8q21.2 from the UCSC browser (GRCh37/hg19). The FISH clones are in green with RP11-90G23 manually annotated from BAC end sequence data (base pairs 86 570 255–86 817 480). The flanking SDs are represented by black double-headed arrows between black vertical lines and have been labelled REPP (for REPeat Proximal) and REPD (for REPeat Distal). The sequence gap between the SDs is represented by the black bar labelled GAP. The RefSeq genes are in blue with the tandem REXO1L1 gene array highlighted by the red arrows underneath. The region contains no non-coding sequences. The DGV tracks show gains (blue), losses (red) and inversions (brown). The UCSC SD track is mostly in terracotta.

Figure 3

(a) Comparison of the total (diploid) copy number of the REXO1L1 gene cluster measured by PFGE Southern blot and the NanoString nCounter Analysis System showing the close linear correlation; (b) distribution of the frequency of REXO1L1 copy numbers (y axis) binned into intervals of 10 copies from 90 to 280 (x axis) in 216 controls. The median copy number of 166 is indicated by the dotted line labelled M, the mean value of 173 by the dashed line labelled A and the copy numbers of 265 and 270 in Patient 4 and her father represented by the black stars.