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. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study did not receive any funding ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The study was approved by the Ethical Committee of the Medical Faculty of the Leipzig University and the authors received and archived written consent of the affected individuals and/or their legal guardians to publish genetic and clinical data as well as photographs. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors