CytoSure® Constitutional v3 arrays offer enhanced exon-level coverage of developmental disorder genes. They combine the up-to-date and relevant developmental delay content from the recent Deciphering Developmental Disorders (DDD) study and latest updates from ClinGen*. The addition of a research-validated collection of single nucleotide polymorphism (SNP) probes with the CytoSure Constitutional v3 +LOH array facilitates the precise identification of loss of heterozygosity (LOH) and uniparental disomy (UPD) in addition to accurate copy number (CN) detection. This offers cost-effective detection of a broader range of genetic syndromes — without the requirement for further investment in equipment or training.
CytoSure Constitutional v3 arrays include probes for up to 502 highly-targeted developmental delay genes identified by both the DDD project and ClinGen, enabling detection of single exon aberrations. Higher probe density across the exons and introns of important developmental delay genes allows improved detection of small (<500bp) deletions and duplications that might otherwise be missed or require manual calling on other outdated constitutional cytogenetics array designs (Figure 1).
Figure 1: Accurate detection of a small, single-exon (<500bp; 4 probes) duplication in MID1 associated with Opitz-G syndrome†. Data generated using the CytoSure Constitutional v3 (8x60k) array.
An informed, sophisticated approach to array design has been used with CytoSure Constitutional v3 arrays, with more probes being located in regions of the genome that are most likely to detect a biologically relevant aberration. These biologically relevant regions have been identified and prioritized during the DDD study and through the ClinGen dosage sensitivity map, with the highest priority regions being covered at an exon-level resolution. In addition, a tiered backbone approach has been adopted with a greater concentration of probes in regions where novel aberrations are more likely to be uncovered, without compromising on overall backbone resolution (Table 1).
Table 1: Selection guide for CytoSure Constitutional v3 arrays.
For a number of years, microarrays have been considered the first approach for research into CNV analysis in children with developmental delay, intellectual disability, and congenital anomalies. The CytoSure Constitutional v3 +LOH array offers the detection of whole chromosome aneuploidies, submicroscopic deletions and duplications and single-exon CNV detection (Figure 1). In addition, the inclusion of empirically selected SNP probes facilitates the detection of copy-neutral events such as LOH, UPD and long-contiguous stretch of homozygosity (LCSH) of 7Mb and above, which can be associated with consanguinity (Figure 2). Additionally, SNPs can provide an internal confirmation of CNVs that may eliminate the need for follow up investigation (Figure 3).
Figure 2: Multiple regions of homozygosity associated with parental consanguinity detected using the CytoSure Constitutional v3 +LOH array. The close-up view illustrates a section of LOH of 7.05Mb on 21q‡.
Figure 3: A t(X;4) unbalanced translocation detected using the CytoSure Constitutional v3 +LOH array. The close-up view of the loss on Chromosome 4 clearly illustrates how the SNP probes act as an independent verification of the copy number change§.
CytoSure Constitutional v3 arrays are available in a range of formats to match your resolution and throughput requirements. All CytoSure arrays have been research-validated using CytoSure Genomic DNA Labelling Kits, which have been uniquely developed and optimized to enable rapid delivery of high-quality results with excellent signal-to noise ratios and superior DLRs. Two formats are available: the CytoSure Genomic DNA Labelling Kit is sufficient for 24 samples and is ideal for labs running one or two arrays a week, and for high-throughput labs, the CytoSure HT Genomic DNA Labelling Kit is recommended as its plate-based protocol allows simultaneous labeling of 96 samples. To achieve the best quality data possible, it is recommended that CytoSure arrays are used in conjunction with CytoSure Genomic DNA Labelling Kits.
As laboratories scale up their processes to increase throughput and reduce costs, parallel processing of higher numbers of samples increases the possibility of sample mix-up. Even automated workflows contain several steps where sample identity can be lost (e.g. pipetting samples into gasket slides). CytoSure Constitutional v3 arrays contain sample tracking probes which, when used in conjunction with CytoSure Sample Tracking Spike-ins, enable researchers to quickly and easily identify any erroneous samples, ensuring only accurate data is reported.
CytoSure Interpret Software, which accompanies all CytoSure arrays, is a powerful, easy-to-use package for the analysis of CNV and SNP data with the ability to analyse data in both hg19 and hg38. Innovative features such as the Accelerate Workflow and automatic aberration classification functionality enable the automation of data analysis workflows, minimizing the need for user intervention and maximizing the consistency and speed of data interpretation.
The unique database enables easy back-up of data, simplified searching and user tracking. CytoSure Interpret Software also includes extensive annotation tracks covering syndromes, genes, exons, benign and pathogenic CNVs. These link to publicly available databases such as ClinGen, Ensembl and the Database of Genomic Variants, providing results in context.
Understanding the complex genetic composition of consanguineous samples, samples with UPD and samples with long stretches of LOH is also simplified using CytoSure Interpret Software. Regions of LOH can be identified by viewing the B-allele frequency (BAF) plot or the Allele Status Plot. CytoSure Interpret Software further simplifies the analysis of LOH using a proprietary ‘LOH Score’ whereby, continuous stretches of homozygous SNPs are scored and those regions with a score above a recommended threshold are considered to be significant (Figure 4).
It is easy to get started as the software is provided with full on-site training. If necessary, legacy array data can also be converted and loaded into CytoSure Interpret Software allowing seamless transfer of aberration information for more powerful analysis.
Figure 4: Chromosome 6 data from a sample where extensive regions of LOH have been detected using the CytoSure Constitutional v3 +LOH array. CytoSure Interpret Software clearly displays the percentage of homozygous alleles.
We have now implemented the OGT CytoSure Constitutional v3 array platform at the West Midlands Regional Genetics Laboratory for the study of developmental disorders in both pre- (foetal malformation) and postnatal samples. The quality of the evidence base behind the array design allows for very comprehensive analysis of the key exons, genes and regions implicated in all known developmental disorders. We found it provided the claimed exon-level resolution and out-performed the other platforms in the ability to detect exon level variants.
Dominic McMullan
Consultant Clinical Scientist, West Midlands Regional Genetics Laboratory, Birmingham Women’s NHS Trust, UK
* Formerly known as ISCA/ICCG. † Data kindly provided by WMRGL Birmingham UK. ‡ Data kindly provided by the University of Louisville, Kentucky, USA. § Data kindly provided by the University of Illinois, Chicago, USA.
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