We have performed whole genome expression and cell function studies in carriers of 1q21.1 deletion and 1q21.1 duplication. Our data show that the top genes ranked based on correlation of expression and 1q21.1 copy number change are significantly enriched for 1q21.1 genes, indicating association of expression and copy number for ~50% of 1q21.1 CNV genes. Furthermore, we show that the function of proteins coded by two of the genes from the 1q21.1 CNV, which ranked highest in 1q21.1 copy number expression correlation, is altered in both the deletion and duplication patient cell lines.
CHD1L, the gene which ranked first in the expression/1q21.1 copy number correlation, has been implicated in chromatin remodeling and relaxation as well as DNA damage response [25, 26]. Our studies identified a novel role for CHD1L in decatenation, which was suspected based on its known chromatin remodeling function, and the defective Topo II decatenation checkpoint demonstrated here in both the 1q21.1 Del and Dup containing patient cell lines.
It is of interest that the DCC defect detected in the 1q21.1 Del and Dup containing cell lines is comparable to that seen in cells from Werner syndrome (OMIM 277700), an autosomal recessive disorder, associated with predisposition to cancer and premature aging, neither of which were noted in our patients. The only overlapping feature, short stature, was noted in 5/6 subjects with the 1q21.1 deletion and also reported in subjects from other cohorts . Previous DCC studies of Werner syndrome and control cells suggested that DCC defect per se is not sufficient to cause significant genomic instability, but requires absence or dysfunction of "caretaker" genes such as ATR, BRCA1 or WRN . It is possible that in cell lines with 1q21.1 Del and Dup the DCC defect is not accompanied with other deleterious events and thus the threshold for significant spontaneous genomic instability leading to premature cell senescence/cancer predisposition is not met. We have not found evidence of spontaneous chromosome instability in the short term chromosome cultures of our patients nor has this been previously reported for any of the1q21.1 CNV subjects who had routine chromosome analysis. Future studies of the association of CHD1L with other genes in decatenation checkpoint mechanism may shed more light on the precise role of CHD1L in DCC. So, whilst the phenotypic consequences of defective DCC activation in subjects with a 1q21 CNV are unclear, their cellular phenotype does appear to be consistent with CHD1L dysfunction.
Our findings that the same cellular phenotype is present in both the 1q2.1 Del and Dup containing cell lines, is in keeping with reports  that both dosage imbalances of CHD1L result in identical cellular effects. Haploinsufficiency and duplication sensitivity is thought to affect genes regulating balanced expression of other genes ("master genes" ), which is in keeping with CHD1L's role as a chromatin remodeler and indirect regulator of many key biological processes such as replication, transcription and translation . In that respect, it is interesting to note that > 18 genes with a role in chromatin remodeling have been implicated in intellectual disability .
PRKAB2, which ranked 10th in the expression/1q21.1 copy number correlation, encodes the β2-subunit of AMPK, a key regulator of cellular response to a large number of external stimuli which modulates energy levels at the cellular and organism level . The deregulation of AMPKβ2 function in 1q21.1 deletion and duplication carriers was suspected based on a) changes in levels of AMPKβ2 protein (in keeping with the 1q21.1 copy number state and expression level of the PRKAB2 gene), b) different basal levels of p-T172-AMPKα in both 1q21.1 Del and Dup containing lines in comparison to WT, and c) sub-optimal AICAR-induced phosphorylation of the AMPK substrates ACC and RAPTOR, which was more obvious in the 1q21.1 Del containing line. The last observation could be explained by the fact that AMPK, as a multi protein complex, may be sensitive to imbalances of its components , and that reduced availability of a regulatory β-isoform, as occurs here, could impact on AMPK activity more than over-abundance.
The multifaceted nature of AMPK role in brain function is of particular interest to the 1q21.1 phenotype which most consistently includes some form of learning difficulty. Previous studies showed that alternations of AMPK activity resulted in profound abnormalities of the central nervous system in AMPK-β1-/- knockout mice which had reduction of AMPK activity , whereas the consequences of AMPK activation remain controversial as some groups have shown that AMPK activation is neuroprotective while others show that AMPK overactivation is detrimental . The essential role of AMPK in brain function is further supported by its inhibition of the mTOR pathway  which is required for learning and memory .
Our studies are the first to report that the function of two genes integral to 1q21.1 CNV is changed in patient lymphoblasts and that both genes are likely to be dosage sensitive. Both genes are expressed in multiple tissues, including brain [34, 41] which may explain the multi-systemic nature of the physical abnormalities, and the frequent involvement of learning difficulty albeit at a very variable levels. It remains uncertain as to the tissue-specific consequences of gene function changes in individuals with 1q21.1 CNV although AMPK is clearly involved in brain development and homeostasis. We believe that our investigations are unique as they pointed to genes for which further functional investigation in additional carriers and cell lines may prove to be worthwhile.
The phenotypic variability for some CNVs has been traditionally explained by genetic and environmental factors . In that respect it is of interest to note that CHD1L and PRKAB2 have a role in sensing and responding to genomic (chromosomal structure) and metabolic (energy level) stress and therefore their dysfunction may result in a more severe phenotype in individuals which experienced more adverse environmental conditions during development. Sequence changes of other genes from the 1q21.1 region as well as other genes across the genome that impair their function cannot be ruled out as a source of variability at this time and the new whole genome sequencing technologies will no doubt become useful in future investigations of their contribution to the development of an abnormal phenotype.