Leveraging Unique Chromosomal Microarray Probes to Accurately Detect Copy Number at the Highly Homologous 15q15.3...

Area of Science:

• Genetics
• Genomic Medicine
• Molecular Diagnostics

Background:

• Biallelic deletions in 15q15.3, encompassing STRC and CATSPER2, cause autosomal recessive deafness-infertility syndrome (DIS).
• Nonsyndromic hearing loss can result from biallelic deletions of STRC alone.
• These deletions are primary genetic drivers of mild-to-moderate hearing loss, yet chromosomal microarray (CMA) detection is hindered by homologous pseudogenes.

Purpose of the Study:

• To evaluate the efficacy of a common CMA platform in detecting copy number variants (CNVs) within the challenging 15q15.3 region.
• To investigate the impact of pseudogene homology on CMA performance for 15q15.3 CNV detection.

Main Methods:

• Twenty-two samples with confirmed 15q15.3 CNVs (verified by droplet digital PCR) were analyzed using CMA.
• Probe-level analysis assessed homology, comparing log2 ratios of unique probes versus pseudogene-homologous probes.
• The impact of pseudogene homology on CMA performance was investigated.

Main Results:

• CMA showed only 40.9% concordance with ddPCR for 15q15.3 CNVs, with frequent zygosity miscalls by automated software.
• High probe homology to pseudogenes significantly contributed to discordance, evidenced by differing log2 ratios.
• Two specific probe clusters with unique sequences reliably detected STRC/CATSPER2 CNVs, achieving 100% concordance with ddPCR, distinguishing homozygous/heterozygous deletions and complex rearrangements.

Conclusions:

• Manual analysis focusing on unique CMA probe clusters, free from significant pseudogene homology, enhances CNV detection and zygosity assignment in the 15q15.3 region.
• Integrating this targeted analysis into CMA workflows can improve diagnostic accuracy for DIS and carrier screening.