Postnatal genome-wide CNV analysis

The analysis is performed using short-read sequencing with Illumina Sequencing-by-Synthesis technology at an average sequencing depth of approximately 7x. Sequencing reads are aligned to the reference genome and assessed bioinformatically for deviations in read depth consistent with copy number loss or gain.

The method detects genome-wide DNA imbalances at an average functional resolution of approximately 10 kb, depending on CNV size, genomic region, mappability, GC content, sequencing quality and bioinformatic confidence.

Detected variant types include whole chromosome aneuploidies, partial chromosomal aneuploidies, terminal and interstitial deletions, terminal and interstitial duplications, recurrent microdeletion and microduplication syndromes, CNVs affecting individual genes, where size and genomic context allow reliable detection.

Because lcWGS analysis is based on sequencing coverage rather than probe hybridisation, it provides a more continuous genome-wide profile than array CGH. This can improve breakpoint definition, particularly in regions with limited array probe coverage.

The assay has been scientifically and bioinformatically validated for postnatal genome-wide CNV detection. Validation data demonstrate sensitivity greater than 95% at an average functional resolution of approximately 10 kb, depending on the genomic region.

During validation, lcWGS-based CNV results showed a high level of concordance with chromosomal microarray findings. Most cases demonstrated identical diagnostic outcomes. Differences were mainly related to breakpoint determination, reflecting the different technical principles of the two methods.

Key analytical strengths:

• Genome-wide CNV detection without probe-bound gaps
• High sensitivity for clinically relevant deletions and duplications
• Detection of partial and complete aneuploidies
• Improved breakpoint delineation in many cases
• Reliable assessment of recurrent and non-recurrent pathogenic CNVs
• Potential detection of intragenic CNVs, depending on size and region

Analytical limitations:

The method is designed to detect unbalanced copy number changes. It generally does not reliably detect balanced translocations, inversions, triploidy or low-level mosaicism. It is not designed as a primary test for single nucleotide variants, small insertions or deletions, methylation abnormalities, repeat expansions or imprinting disorders. For complex cytogenetic questions, supplementary testing may be required.

Postnatal genome-wide CNV analysis is indicated when there is clinical suspicion of a chromosomal imbalance. It is particularly useful in patients with unexplained neurodevelopmental disorders, congenital anomalies or syndromic presentations.

Typical indications include:

• Intellectual disability or global developmental delay
• Autism spectrum disorder
• Multiple congenital anomalies
• Dysmorphic or syndromic features
• Suspected microdeletion or microduplication syndrome
• Suspected partial chromosomal imbalance
• Previously negative karyotyping where a submicroscopic CNV remains possible
• Selected cases with previously negative chromosomal microarray results

Examples of recurrent clinically relevant CNVs

Abbreviations: CNV, copy number variant; Del, deletion; Dup, duplication; lcWGS, low-coverage whole genome sequencing; WGS, whole genome sequencing.

Postnatal genome-wide CNV analysis is appropriate when an unbalanced chromosomal abnormality is suspected, especially in developmental delay, intellectual disability, autism spectrum disorder, multiple congenital anomalies, or dysmorphic or syndromic features.

CNVs are interpreted using recognised professional standards for constitutional CNV classification and reporting, including a five-tier classification: pathogenic, likely pathogenic, variant of uncertain significance, likely benign and benign.

Genetic counselling is recommended to explain possible outcomes, limitations, inheritance, recurrence risk and whether parental or further testing may be indicated. A result with no reportable CNV detected does not exclude a genetic condition.

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