Incorporating Nanopore Sequencing Into a Diverse Diagnostic Toolkit for Incontinentia Pigmenti

Area of Science:

• Genetics
• Molecular Biology
• Human Disease Genetics

Background:

• Incontinentia Pigmenti (IP) is a rare genetic disorder primarily affecting females, with complex genetic analysis due to a homologous pseudogene.
• Conventional sequencing methods struggle to accurately identify variants in the IKBKG gene in IP patients.
• Skewed X-inactivation patterns can assist in variant assignment to the IKBKG gene.

Purpose of the Study:

• To comprehensively identify and phase a heterozygous deletion in the IKBKG gene in an IP-affected family.
• To evaluate the utility of nanopore sequencing in precisely localizing genetic variants and analyzing X-chromosome inactivation status.
• To improve the diagnostic accuracy and understanding of Incontinentia Pigmenti.

Main Methods:

• Whole-exome sequencing (WES) for initial variant detection.
• Long-range PCR and RT-PCR to confirm variant location and RNA expression.
• X-inactivation analysis and nanopore sequencing for precise variant phasing and methylation status assessment.

Main Results:

• A small heterozygous deletion (NM_001099857.5: c.363_367del, p.(Leu122Glyfs*14)) in the IKBKG gene was identified and phased.
• Skewed X-inactivation was observed in both the IP patient and her mother.
• Nanopore sequencing confirmed the variant's location in IKBKG, demonstrated nonsense-mediated decay, and elucidated the skewed X-inactivation pattern.

Conclusions:

• A multi-modal approach combining WES, PCR, and nanopore sequencing is effective for diagnosing IP.
• Nanopore sequencing offers significant value in precise variant localization and X-chromosome inactivation analysis in females.
• Accurate genetic diagnosis and understanding of X-inactivation are crucial for managing IP.