Incorporating Nanopore Sequencing into a Diverse Diagnostic Toolkit for Incontinentia Pigmenti

Background Incontinentia pigmenti (IP) is a rare, hereditary multisystemic disorder affecting 1.2 in 100,000 live births, predominantly females. Conventional genetic analyses through short-read sequencing are complicated in case of IP due to the presence of a highly homologous pseudogene. Traditionally, long-range PCR is employed in order to overcome this challenge, however, detection of skewed X-Inactivation can also aid in correctly assign a variant to IKBKG . Methods We employed a comprehensive multi-method approach, incorporating whole-exome sequencing (WES), long-range PCR, RT-PCR, X-inactivation analysis, and nanopore sequencing of genomic DNA, to identify and accurately phase a small heterozygous deletion NM_001099857.5:c.363_367del, p.(Leu122Glyfs*14) to the IKBKG gene in a family affected with IP. Results The deletion was initially detected through WES and skewed X-inactivation was observed in both the proband and her mother. Long-range PCR specific to IKBKG was utilized to verify that the variant is located in IKBKG and not in its highly homologous pseudogene. On RNA level, the variant was undetectable, suggesting nonsense-mediated decay (NMD) of the transcript containing the variant. We further utilized nanopore sequencing not only to pinpoint and accurately map the variant to the IKBKG gene but also to analyze methylation status of both alleles. This allowed us to confirm the skewed X-inactivation, with the variant-carrying allele found to be predominantly inactivated. Conclusion Nanopore sequencing serves as a valuable tool in genetic diagnosis, enabling the precise localization of the variant in either the gene or the pseudogene. Furthermore, in females with skewed X-inactivation, this method facilitates the determination of whether the variant is predominantly located on the activated or inactivated X-chromosome.