Incorporating Nanopore Sequencing Into a Diverse Diagnostic Toolkit for Incontinentia Pigmenti

Abstract

Incontinentia pigmenti (IP) is a rare hereditary disorder affecting 1.2 in 100,000 live births, predominantly females. Genetic analysis of IP is complicated by a homologous pseudogene, making conventional short-read sequencing challenging. While long-range PCR is typically used to overcome this, skewed X-inactivation detection can also aid in assigning variants to IKBKG. We employed a comprehensive approach, incorporating whole-exome sequencing (WES), long-range PCR, RT-PCR, X-inactivation analysis, and nanopore sequencing, to identify and accurately phase a small heterozygous deletion, NM_001099857.5: c.363_367del, p.(Leu122Glyfs⁣^∗14), in the IKBKG gene in an IP-affected family. The deletion was initially detected via WES, with skewed X-inactivation observed in both the proband and her mother. Long-range PCR specific to IKBKG confirmed the variant's location in the IKBKG gene, not in the pseudogene. On the RNA level, the variant was undetectable, suggesting nonsense-mediated decay of the transcript. Nanopore sequencing precisely mapped the variant to IKBKG and analyzed the methylation status of both alleles, confirming the skewed X-inactivation, with the variant-carrying allele predominantly inactivated. This demonstrates the nanopore sequencing's value in genetic diagnosis, enabling precise variant localization and analysis of X chromosome activation status in females with skewed X-inactivation, aiding in accurate diagnosis and understanding of IP.

Figure 1

The IKBKG locus on chromosome X (adapted from Pescatore et al. [12]). (a) Schematic representation of the Xq28 locus including IKBKG, its pseudogene IKBKGP1, and the NM_001099857.5 transcript. The square grey arrow boxes represent the low copy repeats (LCR1/2) of the segmental duplication, the exons of IKBKG/IKBKGP1 are depicted in blue, and the exons of G6PD in red. The IKBKG gene incorporates nine coding exons and four alternative noncoding exons (1A–1D) that overlap with the locus of G6PD. Translation initiation site is located at the beginning of Exon 2, marked here with ORF. The transcript is composed of the Exons 2–10; the identified variant c.363_367del in Exon 3 of IKBKG is marked with a red triangle. RNA primer pairs used for confirmation of variant location are shown as green and orange arrows (primer pair B = orange arrows, primer pair C = green arrows; see Table S2). (b) Schematic representation of the IKBKG protein, its domains as well as indication of the identified variant p.(Leu122Glyfs⁣^∗14) in NP_001093327 (419 aa). Domains are as follows: HLX1: helical domain; CC1: coiled coil; HLX2: helical domain; CC2: coiled coil; NUB: NEMO ubiquitin binding; LZ: leucin zipper; ZF: zinc finger; aa ranges are indicated below and above the domain.

Figure 2

Individuals' skin manifestations. (a–c) Skin blisters on the upper and lower extremities and face. (d) Healed skin on the left leg at the age of 18 months, faint white maculae visible.

Figure 3

IGV screenshot of aligned short-read whole-exome sequencing reads and nanopore reads: The genomic region around the variant c.363_367del, p.(Leu122Glyfs⁣^∗14) in the IKBKG gene (left) as well as the homologous region in the pseudogene IKBKGP1 (right) in which none of the nanopore reads show the variant of interest. Nanopore reads are colored according to haplotypes. Transparent reads in WES data specify mapping quality of 0 indicating that reads cannot be mapped conclusively to a single region.

Figure 4

Haplotype-specific methylation profiles of the IKBKG locus showing increased methylation of the variant-carrying maternal allele (Haplotype 2, orange) compared to the wild-type paternal allele (Haplotype 1, blue). From top to bottom, this plot shows a gene track, haplotype-specific methylation calls (black color coded) relative to aligned read positions, a translation from genome space into a modified base space consisting only of instances of the methylated motif, the haplotype-specific methylation statistic (log-likelihood ratio—1: likely methylated, 0: likely unmethylated), and a smoothed sliding window plot showing the methylated fraction across the region (IKBKG gene ± 100 kb). The promoter region of the IKBKG gene is highlighted in light blue.