Detection of revertant mosaicism in epidermolysis bullosa through Cas9‐targeted long‐read sequencing

Natsuga, Ken; Furuta, Yoshikazu; Takashima, Shota; Nohara, Takuma; Kosumi, Hideyuki; Mai, Yosuke; Higashi, Hideaki; Ujiie, Hideyuki

doi:10.1002/humu.24331

Cited by 6 publications

(5 citation statements)

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“…At the time of the initial description of RM in a genodermatosis in 1997, RM was considered an extraordinary phenomenon. However, since that initial description, RM has been identified in all major types of EB (Table 1) [ 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 ], and, in 2012, we were able to confirm revertant skin patches in all ten Dutch patients with the intermediate type of JEB due to pathogenic COL17A1 variants [ 79 ]. Around the same time, Choate et al demonstrated that each of the multiple healthy ‘confetti-like’ spots in patients with ichthyosis with confetti (IWC) due to germline variants in KRT10 (IWC-I) or KRT1 (IWC-II) represent a separate occurrence of RM in a single keratinocyte clone [ 80 , 81 ].…”

Section: Revertant Mosaicism In Genodermatosesmentioning

confidence: 86%

“…Another class of reversion mechanisms involves chromosomal recombination events during mitosis, i.e., gene conversion, intragenic cross-overs, and mitotic recombination. Only a few correction mechanisms identified in EB involve such recombination events [ 26 , 64 , 65 , 70 , 72 , 82 , 86 ], probably because most EB patients in which RM has been identified have biallelic gene mutations where recombination events are less likely to create cells without mutations. In contrast, mitotic recombination is the only reversion mechanism so far identified in the autosomal dominant disorders IWC-I, IWC-II, LK, and PRP [ 80 , 81 , 83 , 84 , 88 , 89 , 90 , 91 ].…”

Section: Molecular Mechanisms Of Revertant Mosaicism In Genodermatosesmentioning

confidence: 99%

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Revertant Mosaicism in Genodermatoses: Natural Gene Therapy Right before Your Eyes

2022

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Revertant mosaicism (RM) is the intriguing phenomenon in which nature itself has successfully done what medical science is so eagerly trying to achieve: correcting the effect of disease-causing germline variants and thereby reversing the disease phenotype back to normal. RM was molecularly confirmed for the first time in a genodermatosis in 1997, the genetic skin condition junctional epidermolysis bullosa (EB). At that time, RM was considered an extraordinary phenomenon. However, several important discoveries have changed this conception in the past few decades. First, RM has now been identified in all major subtypes of EB. Second, RM has also been identified in many other genodermatoses. Third, a theoretical mathematical exercise concluded that reverse mutations should be expected in all patients with a recessive subtype of EB or any other genodermatosis. This has shifted the paradigm from RM being an extraordinary phenomenon to it being something that every physician working in the field of genodermatoses should be looking for in every patient. It has also raised hope for new treatment options in patients with genodermatoses. In this review, we summarize the current knowledge on RM and discuss the perspectives of RM for the future treatment of patients with genodermatoses.

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Section: Revertant Mosaicism In Genodermatosesmentioning

confidence: 86%

Section: Molecular Mechanisms Of Revertant Mosaicism In Genodermatosesmentioning

confidence: 99%

Revertant Mosaicism in Genodermatoses: Natural Gene Therapy Right before Your Eyes

2022

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“…These sequencers typically generate long to ultra‐long reads. A combination of CRISPR/Cas9 technology and nanopore sequencing has been further shown to enable enrichment and sequencing of specific regions in genomic DNA (gDNA) (Gilpatrick et al, 2020) without PCR procedures or reverse transcription procedures, which can induce artificial homologous recombination (Meyerhans et al, 1990; Natsuga et al, 2022; Negroni et al, 1995). This method is called nanopore Cas9‐targeted sequencing (nCATS) and has been utilized to detect somatic mutations and chromosomal rearrangements (Gilpatrick et al, 2020; Natsuga et al, 2022; Stangl et al, 2020; Watson et al, 2020; Wongsurawat et al, 2020).…”

Section: Figurementioning

confidence: 99%

“…Although regular genetic testing might not always need nCATS, this method can be an option for haplotyping somatic mutations (Natsuga et al, 2022), haplotyping three or more variants in one patient like our study, and identifying repeat expansions (Mizuguchi et al, 2021;Sone et al, 2019). One nCATS experiment costs hundreds of USD, mostly for a MinION flow cell.…”

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confidence: 99%

Cas9‐guided haplotyping of three truncation variants in autosomal recessive disease

et al. 2022

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An autosomal recessive disease is caused by biallelic loss-of-function mutations.However, when more than two disease-causing variants are found in a patient's gene, it is challenging to determine which two of the variants are responsible for the disease phenotype. Here, to decipher the pathogenic variants by precise haplotyping, we applied nanopore Cas9-targeted sequencing (nCATS) to three truncation COL7A1 variants detected in a patient with recessive dystrophic epidermolysis bullosa (EB).The distance between the most 5′ and 3′ variants was approximately 19 kb at the level of genomic DNA. nCATS successfully demonstrated that the most 5′ and 3′ variants were located in one allele while the variant in between was located in the other allele. Interestingly, the proband's mother, who was phenotypically intact, was heterozygous for the allele that harbored the two truncation variants, which could otherwise be misinterpreted as those of typical recessive dystrophic EB. Our study highlights the usefulness of nCATS as a tool to determine haplotypes of complicated genetic cases. Haplotyping of multiple variants in a gene can determine which variant should be therapeutically targeted when nucleotide-specific gene therapy is applied.

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“…As most recessively inherited EB carries compound heterozygous mutations, confirmation of RM is necessary before expansion. Identification of RM is challenging, but recently a method has been proposed based on Cas9 enrichment and long-run nanopore sequencing that identify revertant correction resulting from homologous recombination (Natsuga et al 2022). The genetic mechanism behind this phenomenon remains uncertain but it has been associated with back mutations, mitotic recombination, and novel mutations during cell division (Kiritsi et al 2014).…”

Section: Spontaneous Revertant Mosaicismmentioning

confidence: 99%

Stairways to Advanced Therapies for Epidermolysis Bullosa

Rosa

Enzo

Palamenghi

et al. 2022

Cold Spring Harb Perspect Biol

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Epidermolysis bullosa (EB) is a devastating genetic skin disease typified by a plethora of different phenotypes and ranking from severe, early lethal, to mild localized forms. Although there is no cure for EB, recent progress in pharmacology and molecular and cellular biology is boosting the development of new advanced therapeutic strategies. Here we will focus on two main categories of such therapies: (1) those aimed at controlling inflammation and inducing reepithelialization of the wounds, and (2) those, perhaps more challenging and ambitious, that aim to permanently regenerate a fully functional epidermis, which requires targeting of epidermal stem cells. In both cases, the genetic variants underlying the different EB forms and factors, such as genetic background, modifier genes, comorbidities, and lifestyle, all of which impinge on EB genotype-phenotype correlation, need to be defined.I nherited epidermolysis bullosa (EB) is characterized by recurrent blistering of stratified epithelia. EB is caused by more than 1000 mutations in at least 16 structural genes encoding proteins forming hemidesmosomes and anchoring fibrils, which are essential for the integrity of the epidermal-dermal junction (Has et al. 2020a). Blisters arise spontaneously or upon minimal trauma as the result of the fragility of skin and mucous membranes. Common features of many EB forms include damage of ocular surface, upper airways, oral mucosa and gastrointestinal and renal systems, as well as hair, nail, and enamel defects (Bardhan et al. 2020;Has et al. 2020a). Incidence and prevalence of EB in the United States are 11.1 per one million people and 19.6 per one million live births, respectively. Similar values have been reported in Europe (Fine 2010;Has et al. 2020a). EB affects individuals from all ethnic origins regardless of gender and displays either dominant or recessive patterns of inheritance. EB phenotypes range from mild, localized blistering to massive blistering, erosions and chronic wounds. Severe EB forms can be early lethal and generalized EB frequently leads to aggressive squamous cell carcinoma (SCC) (Condorelli et al. 2019;Bardhan et al. 2020).

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Detection of revertant mosaicism in epidermolysis bullosa through Cas9‐targeted long‐read sequencing

Cited by 6 publications

References 30 publications

Revertant Mosaicism in Genodermatoses: Natural Gene Therapy Right before Your Eyes

Revertant Mosaicism in Genodermatoses: Natural Gene Therapy Right before Your Eyes

Cas9‐guided haplotyping of three truncation variants in autosomal recessive disease

Stairways to Advanced Therapies for Epidermolysis Bullosa

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