Date of Award

January 2020

Document Type

Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Keith A. Choate

Abstract

Introduction

Genetic mosaicism underlies many inherited and acquired cutaneous disorders. Despite the barriers to genetic analysis of mosaic disorders, next generation sequencing has led to substantial progress in understanding their pathogenesis, which has significant implications for clinical management and genetic counselling. Advances in paired and deep sequencing technologies in particular have made study of mosaic disorders more feasible. In this study, we aim to utilize next generation sequencing technologies in order to evaluate the genetic cause of three mosaic cutaneous disorders: epidermolytic acanthoma (EA), progressive symmetric erythrokeratodermia (PSEK), and mosaic childhood acantholytic dyskeratosis.

Methods

Using genomic DNA extracted from paraffin-embedded samples from departmental archives of EA, we evaluated a discovery cohort using whole-exome sequencing (WES) and assessed remaining samples using Sanger sequencing screening and restriction fragment length polymorphism (RFLP) analysis. We analyzed blood and saliva DNA from cases of PSEK referred from academic dermatologists via WES and verified mutations via Sanger sequencing. We isolated and cultured keratinocytes from a case of mosaic childhood acantholytic dyskeratosis and performed paired RNA sequencing in comparison to blood RNA.

Results

DNA from 16/20 cases of EA in our sample was of sufficient quality for PCR amplification. WES of genomic DNA from lesional tissue revealed KRT10 c.466C>T, p.Arg156Cys mutations in 2/3 samples submitted for examination. RFLP analysis of these samples, as well as 8 additional samples confirmed the mutations identified via WES and identified 4 additional cases with Arg156 mutations. In sum, 6/11 screened cases of EA demonstrated hotspot mutation in KRT10. Additionally, all three cases of progressive symmetric erythrokeratodermia had compound heterozygous mutations of the ABCA12 gene. One case had missense mutations on both copies of the ABCA12 gene (c.2531A>G, p.D844G; c.5812C>T, p.P1938S). Another case had a nonsense mutation on one copy of ABCA12 (c.5787T>G, p.Y1929*) and a missense mutation on the other (c.6852G>C, p.E2284D). A third case had a frameshift mutation (c.2033_2036delATCA, p.N678Rfs*10) on one copy of ABCA12 and a splice site mutation (c.2866-8T>A) on the other. Finally, paired RNA sequencing of cultured keratinocytes from lesional tissue of a case of mosaic acantholytic dyskeratosis revealed a somatic mutation in GJB2 (c.T83A, p.L28H) which was absent in both of the patient's parents.

Conclusions

In this work, we present evidence for the genetic mechanism of three mosaic cutaneous conditions. Our findings of hotspot mutations in the Arg156 position of KRT10 in EA support the hypothesis that it is a somatic mosaic form of epidermolytic ichthyosis. Our finding of ABCA12 mutations in PSEK combined with the involvement of ABCA12 in ceramide transport contributes to a growing body of evidence for the role of ceramide dysfunction in the pathogenesis of PSEK. Finally, we present evidence that mosaic KID syndrome may present with acantholytic dyskeratosis.

Comments

This thesis is restricted to Yale network users only. It will be made publicly available on 09/10/2022

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