Advanced phasing techniques in congenital skin diseases

Abstract

Phasing, the process of determining which alleles at different loci on homologous chromosomes belong together on the same chromosome, is crucial in the diagnosis and management of autosomal recessive diseases. Advances in long-read sequencing technologies have significantly enhanced our ability to accurately determine haplotypes. This review discusses the application of low-coverage long-read sequencing, nanopore Cas9-guided long-read sequencing, and adaptive sampling in phasing, highlighting their utility in complex clinical scenarios. Through clinical vignettes, we explore the importance of phasing in gene therapy design for recessive dystrophic epidermolysis bullosa and the role of revertant mosaicism in therapeutic epidermal autografts. Despite its promise, phasing with long-read sequencing faces challenges, including low efficiency in enriching target regions and the inherent error rate of nanopore sequencing. Future developments in long-read sequencing technologies will be critical in overcoming these limitations and expanding the applicability of phasing across various clinical settings.

Keywords: epidermolysis bullosa; long‐read sequencing; nanopore sequencing; revertant mosaicism.

FIGURE 1

Basics of phasing in autosomal recessive diseases. (a) Cis and trans of two variants, depicted as magenta and blue rectangles. (b) Phasing by inferring through the results of paternal DNA samples. (c) Difficulties in phasing when parental DNA samples are unavailable. (d) Phasing through sequencing when the read length is longer than the distance between the variants. (e) Failure in phasing when the read length is shorter than the distance between the variants. (f) Phasing using another variant (yellow rectangle) by sequencing with a read length that is shorter than the distance between the original two variants (magenta and blue rectangles). (g) Artificial recombination due to a high number of PCR cycles. (h) Nanopore Cas9‐guided long‐read sequencing (nCATS). (i) Nanopore sequencing with adaptive sampling. Nanopore sequencing icon by DBCLS (https://togotv.dbcls.jp/en/pics.html) is licensed under CC‐BY 4.0 Unported (https://creativecommons.org/licenses/by/4.0/).

FIGURE 2

Phasing in glycogen storage disease type Ia using low‐coverage long‐read sequencing. (a) Pedigree of the family with glycogen storage disease type Ia. (b) Sanger sequencing results of the pedigree. (c) Possible diplotypes. (d) Large deletion detected by nanopore sequencing (four reads). Images (a, b, d) reproduced, under CC‐BY 4.0 (https://creativecommons.org/licenses/by/4.0/), from Ref. [9].

FIGURE 3

Phasing in localized porokeratosis using a hybrid strategy. (a) Localized porokeratosis with FDFT1 methylation and genetic variants. (b) Phasing using a combination of amplicon deep sequencing, next‐generation sequencing (NGS), and PacBio low‐coverage long‐read sequencing (2–11 reads). The methylation and genetic variants were present in trans. Images reconstructed from the data on Individual 8, Lesion 4, from Ref. [16].

FIGURE 4

Phasing of three premature termination codon (PTC)‐causing variants in COL7A1 using nCATS. (a) A recessive dystrophic epidermolysis bullosa (RDEB) patient who showed blisters and erosions at birth. (b) Three heterozygous PTC‐causing COL7A1 variants were found in the patient. nCATS strategy and possible diplotypes are shown. (c) Results of nCATS from the proband and mother. (d) Phasing results from nCATS. (e) A strategy of targeted gene therapy for the proband. (f) Possible misdiagnosis of the mother as having RDEB. Images reproduced and modified with permission from Ref. [19].

FIGURE 5

Phasing on revertant mosaicism in recessive dystrophic epidermolysis bullosa using nCATS. (a) Another RDEB patient who was compound heterozygous for c.5932C>T (p.Arg1978Ter) and c.8029G>A (p.Gly2677Ser) in COL7A1. A clinically revertant spot is marked with a dotted line. (b) nCATS strategy. (c) Possible haplotypes. (d) nCATS results. (e) Intragenic crossover in the revertant epidermis. Images reproduced and modified with permission from Ref. [4].

FIGURE 6

Phasing in ichthyosis vulgaris using adaptive sampling. Sixteen ichthyosis vulgaris patients with two heterozygous FLG variants were analyzed. Nanopore sequencing with adaptive sampling showed that all variants were present in trans for both mild and severe cases. Images reconstructed from Ref. [30].