Understanding the impact of 1q21.1 copy number variant

Abstract

Background: 1q21.1 Copy Number Variant (CNV) is associated with a highly variable phenotype ranging from congenital anomalies, learning deficits/intellectual disability (ID), to a normal phenotype. Hence, the clinical significance of this CNV can be difficult to evaluate. Here we described the consequences of the 1q21.1 CNV on genome-wide gene expression and function of selected candidate genes within 1q21.1 using cell lines from clinically well described subjects.

Methods and results: Eight subjects from 3 families were included in the study: six with a 1q21.1 deletion and two with a 1q21.1 duplication. High resolution Affymetrix 2.7M array was used to refine the 1q21.1 CNV breakpoints and exclude the presence of secondary CNVs of pathogenic relevance. Whole genome expression profiling, studied in lymphoblast cell lines (LBCs) from 5 subjects, showed enrichment of genes from 1q21.1 in the top 100 genes ranked based on correlation of expression with 1q21.1 copy number. The function of two top genes from 1q21.1, CHD1L/ALC1 and PRKAB2, was studied in detail in LBCs from a deletion and a duplication carrier. CHD1L/ALC1 is an enzyme with a role in chromatin modification and DNA damage response while PRKAB2 is a member of the AMP kinase complex, which senses and maintains systemic and cellular energy balance. The protein levels for CHD1L/ALC1 and PRKAB2 were changed in concordance with their copy number in both LBCs. A defect in chromatin remodeling was documented based on impaired decatenation (chromatid untangling) checkpoint (DCC) in both LBCs. This defect, reproduced by CHD1L/ALC1 siRNA, identifies a new role of CHD1L/ALC1 in DCC. Both LBCs also showed elevated levels of micronuclei following treatment with a Topoisomerase II inhibitor suggesting increased DNA breaks. AMP kinase function, specifically in the deletion containing LBCs, was attenuated.

Conclusion: Our studies are unique as they show for the first time that the 1q21.1 CNV not only causes changes in the expression of its key integral genes, associated with changes at the protein level, but also results in changes in their known function, in the case of AMPK, and newly identified function such as DCC activation in the case of CHD1L/ALC1. Our results support the use of patient lymphoblasts for dissecting the functional sequelae of genes integral to CNVs in carrier cell lines, ultimately enhancing understanding of biological processes which may contribute to the clinical phenotype.

Figure 1

Comparison of genomic overlap for 1q21.1 CNVs. CNV breakpoints were determined using Affymetrix 2.7 M whole genome array for all subjects except B2 whose breakpoints were determined using a SignatureChip WG v1.1. Red bars indicate deletion of 1q21.1 region while blue bars indicate a duplication. The previously reported minimal deletion region is shown in green. Genes seen in the majority of our cases (core genes) are highlighted in yellow.

Figure 2

Correlation of expression and copy number for probes from chromosome 1 expressed as log₁₀ of the p values (see Methods). The probes from 1q21.1 region are in black.

Figure 3

Functional assays for CHD1L in patient cells. (A) Left panels: Western analysis of CHD1L expression from wild-type (WT), 1q21.1 deletion (Del) and duplication (Dup) LBCs following titration of whole cell extracts. Right Panels: β-tubulin re-probe to confirm equal loading. (B). Densiometric quantification of CHD1L expression from Western analysis from low (black bar), intermediate (white) to higher (grey) amounts of protein, from each line, using three separate determinations, normalized to β-tubulin loading (a.u. arbitrary units). p = 0.009 for Del and p < 0.005 for Dup compared to WT. (C). The Decatenation Checkpoint (DCC). Unreplicated DNA sequences between converging replication forks undergo catenation and torsional tension which is normally relieved by Topoisomerase IIα (Topo IIα) which induces a transient DSB enabling decatenation (untangling). DCC activation in G2 prevents entry into mitosis until sister chromatids are fully separated. DCC can be activated by Topo II inhibitors arresting the cycle in G2 (indicated in red). DCC failure is monitored by the enumeration of 'pseudomitosis' containing highly catenated (entangled) chromatids. Inset image shows typical pseudomitotic cells following treatment of the Del LBCs with the Topo II inhibitor, ICRF-193. (D). Mitotic index (Mitosis %) and Pseudomitotic index (Pseudomitosis %) for untreated (Unt) LBCs or ICRF-193 treated, for wild-type (WT), Werner's syndrome (WRN), Dup and Del containing LBCs. WRN LBCs are known to be defective in DCC activation. Data presented indicates the mean ± s.d of three separate determinations. p < 0.005 for reduction in Mitosis (%) Unt compared to ICRF-193 and p < 0.005 for increase in Pseudomitosis (%) Unt compared to ICRF-193.

Figure 4

Consequences of limiting CHD1L levels by siRNA. (A). Careful titration of CHD1L siRNA conditions were undertaken in A549 so as to mimic haploinsufficient expression of CHD1L. Left panels:These show the Western analysis of whole cell extracts from Untreated (Unt; mock-treated) control whereas siRNA indicates cells treated with CHD1L siRNA. β-tubulin expression was monitored to confirm equal loading. Right graph: Densiometric quantification of CHD1L expression, normalized to β-tubulin loading from three separate siRNA experiments. The degree of CHD1L reduction is very similar to that observed from the 1q21.1 deletion (Del) containing LBC (Fig 3A and B). Data represents the mean ± s.d. of three separate experiments (a.u. arbitrary units). (B). Inset image shows a typical catenated pseudomitotic cell following CHD1L siRNA-mediated knockdown in A549 treated with Topo II inhibitor (ICRF-193). The % pseudomitosis were enumerated under various conditions in A549 following CHD1L siRNA-mediated knockdown to mimic haploinsufficiency. Unt (untreated; not treated with ICRF-193), ICRF-193 (treated with ICRF-193), Con (control siRNA scrambled oligo), CHD1L siRNA (treated with siRNA to mimic CHD1L haploinsufficiency). Data represents the mean ± s.d. of three separate experiments and p < 0.005 for increase in Pseudomitosis (%) following CHD1L siRNA. (C). Inset image shows micronuclei (MN). The % of ICRF-193-induced MN in binucleate cells were determined in wild type (WT), Dup and Del containing LBLs following a 24 hr recovery from an overnight treatment with ICRF-193. Data represents the mean ± s.d. of three separate experiments and p = 0.02 for increase % MN in binucleates for Dup and p < 0.005 for Del containing LBCs.

Figure 5

Functional assays for PRKAB2 in patient cells. (A). Left panels:Titrated whole cell extracts wereblotted for AMPKβ2 (encoded by PRKAB2) in wild-type (WT),Del and Dup containing LBCs. Right panels:Blots were re-probed with anti-β-tubulin. Graph: Densiometric quantification of AMPK-β2 expression from titrated extracts, going from low (black bar), intermediate (white) to higher (grey) amounts of protein, normalized to β-tubulin loading, from three separate determinations (a.u. arbitrary units). p = 0.01 for Del and p < 0.005 for Dup LBCs compared to WT. (B). AMPK subunit AMPK-β1, encoded by the PRKAB1 gene on chromosome 12q24.1, shows equivalent expression in the wild-type (WT), Del and Dup containing LBCs. β-tubulin was used to confirm equal loading. (C). AICAR-induced (2 mM) activation of the AMPK kinase was monitored using phosphorylation of the AMPKα subunit on threonine 172 (p-T172-AMPKα). Dup and Del containing LBCs exhibited higher levels of p-T172-AMPKα at the 0 time (untreated), relative to wild-type (WT). Only the 1q21.1 Del containing LBCs appeared to be unresponsive to AICAR-treatment here. Blots were re-probed with for native AMPKα to confirm loading. (D). AICAR-induced (2 mM) activation of AMPK was evaluated by monitoring phosphorylation of the AMPK substrate Acetyl-CoA Carboxylase on serine 79 (p-S79-ACC). Similar to p-T172-AMPKα, the Del containing LBCs appear unresponsive to the AICAR treatment. Blots re-probed for native ACC to confirm loading. (E). AICAR-induced activation of AMPK was also evaluated by phosphorylation of the AMPK substrate RAPTOR on serine 792 (p-S792-RAP) under identical conditions as in (B) and (C). Again, Del containing LBCs appeared somewhat unresponsive to AICAR. Blots re-probed for MCM2 to confirm loading.