Ultrasensitive profiling of UV-induced mutations identifies thousands of subclinical facial tumors in tuberous sclerosis complex
Katarzyna Klonowska, … , Joel Moss, David J. Kwiatkowski
Katarzyna Klonowska, … , Joel Moss, David J. Kwiatkowski
Published March 31, 2022
Citation Information: J Clin Invest. 2022;132(10):e155858. https://doi.org/10.1172/JCI155858.
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Clinical Research and Public Health Dermatology Genetics

Ultrasensitive profiling of UV-induced mutations identifies thousands of subclinical facial tumors in tuberous sclerosis complex

Abstract

Background Tuberous sclerosis complex (TSC) is a neurogenetic syndrome due to loss-of-function mutations in TSC2 or TSC1, characterized by tumors at multiple body sites, including facial angiofibroma (FAF). Here, an ultrasensitive assessment of the extent and range of UV-induced mutations in TSC facial skin was performed.Methods A multiplex high-sensitivity PCR assay (MHPA) was developed, enabling mutation detection at extremely low (<0.1%) variant allele frequencies (VAFs).Results MHPA assays were developed for both TSC2 and TP53, and applied to 81 samples, including 66 skin biopsies. UV-induced second-hit mutation causing inactivation of TSC2 was pervasive in TSC facial skin with an average of 4.8 mutations per 2-mm biopsy at median VAF 0.08%, generating more than 150,000 incipient facial tumors (subclinical “micro-FAFs”) in the average TSC subject. The MHPA analysis also led to the identification of a refined UV-related indel signature and a recurrent complex mutation pattern, consisting of both a single-nucleotide or dinucleotide variant and a 1- to 9-nucleotide deletion, in cis.Conclusion TSC facial skin can be viewed as harboring a patchwork of clonal fibroblast proliferations (micro-FAFs) with indolent growth, a small proportion of which develop into clinically observable FAF. Our observations also expand the spectrum of UV-related mutation signatures.Funding This work was supported by the TSC Alliance; the Engles Family Fund for Research in TSC and LAM; and the NIH, National Heart, Lung, and Blood Institute (U01HL131022-04 and Intramural Research Program).

Authors

Katarzyna Klonowska, Joannes M. Grevelink, Krinio Giannikou, Barbara A. Ogorek, Zachary T. Herbert, Aaron R. Thorner, Thomas N. Darling, Joel Moss, David J. Kwiatkowski

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Figure 2

MHPA strategy.

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MHPA strategy. (A) Schematic representation of the major MHPA steps. MHP...
(A) Schematic representation of the major MHPA steps. MHPA consists of PCR amplification of short DNA segments using primers that include unique molecular identifier (UMI) barcodes, followed by library preparation and sequencing. In the first reaction (step 1), a multiplex linear amplification of each genomic region occurs using reverse primers only with inclusion of a random 14-nt UMI. Following purification, another linear amplification (step 2) of each genomic region occurs using forward primers only. Following purification, amplification of the UMI-barcoded molecules occurs using universal primers (UP) (steps 3 and 4). Step 3 is performed for optimization of MHPA assays, in which amplicons are labeled using a fluorescent dye, 6-carboxyfluorescein (FAM), followed by capillary electrophoresis to assess abundance of each amplicon. Step 4 is used to generate the MHPA libraries, which are purified and subjected to MPS. (B) Comparison of conventional and UMI-based MPS variant calling strategies. Barcoding of single DNA molecules with UMIs enables compression of the MHPA data to consensus reads that permits sequencing error suppression. (C) Map of deleterious germline TSC2 variants reported in the LOVD database. The y axis indicates the number of TSC2 variants at a single nucleotide position. Recurrent variants appear as vertical lines. The color of the line indicates the type of variant, as shown in the inset legend. Hotspots with variants reported more than 30 times are labeled with coding sequence nucleotide (c.) and amino acid (p.) position. Splice mutations are summed and shown as a single bar at each exon-exon junction. Genomic regions covered by MHPA amplicons are marked in gray, with indication of the fraction of the deleterious germline variants covered by each of the amplicons (%).