Long-read sequencing is required for precision diagnosis of incontinentia pigmenti

Abstract

Incontinentia pigmenti (IP) is caused by loss-of-function variants in IKBKG, with molecular genetic diagnosis complicated by a pseudogene. We describe seven individuals from three families with IP but negative clinical genetic testing in whom long-read sequencing identified causal variants, including one family with the common exon 4-10 deletion not identified by conventional clinical genetic testing. Concurrent methylation analysis explained disease severity in one individual who died from neurologic complications, identified a mosaic variant in an individual with an atypical presentation, and confirmed skewed X chromosome inactivation in an XXY individual.

Keywords: IKBKG; incontinentia pigmenti; long-read sequencing; methylation; pseudogene; skewed X chromosome inactivation; structural variation.

Figure 1

A deletion of IKBKG exons 8–10 likely resulted from an inversion (A) The neonate displayed erythematous papules and vesicles in a Blaschkoid pattern on the abdomen, legs, and arms, consistent with the early findings of IP. (B) Pedigree consistent with X-linked inheritance of an XY male-lethal disorder. The neonate’s mother, maternal aunt, and grandmother had received a clinical diagnosis of IP, which was also suspected in the maternal great-grandmother and great-great-grandmother given their clinical history, recurrent miscarriages, and predominantly XX female offspring. (C) Structure of the IKBKG locus showing the position of IKBKG, two large segmental duplications, and the pseudogene IKBKGP1, which contains nearly identical copies of IKBKG exons 3–10. (D) The deletion identified in the family likely arose via a two-step mechanism. First, an unequal exchange event between Alu transposable elements (triangles) at IKBKGP1 removed exons 8–10 of the pseudogene. Second, an exchange event between the Alu in intron 7 of IKBKG and the remaining Alu in IKBKGP1 resulted in an inversion of the locus, which moved exons 8–10 of IKBKG to IKBKGP1. (E) Phased LRS data shows skewed X-inactivation over a CpG island within IKBKG (box) in the mildly affected mother (IV:1) but random X-inactivation in the more severely affected proband (V:1). Methylation status at CpG islands directs the expression of the gene at that site; methylated (inactive) CpG sites are shown in red and unmethylated (active) CpG sites in blue. The familial exon 8–10 deletion is also indicated (dashed box). Numbers indicate IKBKG exons.

Figure 2

Disease-causing variation missed by prior clinical genetic testing in two families with IP (A–D) Family 2. (A) The proband (II:1) displayed a rash consistent with IP at birth. (B) The proband was the only affected individual in the family; thus, a de novo variant was suspected. (C) Phased IGV view of LRS data with relevant gene regions shown. An atypical inversion bisects IKBKG and GAB3 (dashed boxes) on the paternal haplotype (2). Arrows indicate reads that span from the promoter region of IKBKG to the inversion breakpoint within IKBKG and show that these reads are methylated. The inversion breakpoint within an intron of GAB3 creates a small duplication, leaving one intact copy of GAB3 on the affected haplotype. (D) Subway plot demonstrates the structure of the complex SV, which includes the inversion and duplication. (E–G) Family 3. (E) Multiple females from family 3 with clinical diagnoses of IP also had miscarriages. The proband (III:1) had a mildly affected male sibling who was found to be 47,XXY. (F) A photo of the proband’s left hand shows abnormal nail beds. (G) Analysis of LRS data from individual III:3 (shown) as well as III:1 (not shown) revealed the common exon 4–10 11.7-kbp deletion and skewed X-inactivation. Some reads in individual III:3 carrying the deletion are assigned to haplotype 1 as a result of poor phasing of shorter fragments generated using DNA isolated from saliva.