Mitotic Recombination in Patients with Ichthyosis Causes Reversion of Dominant Mutations in KRT10

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Science  01 Oct 2010:
Vol. 330, Issue 6000, pp. 94-97
DOI: 10.1126/science.1192280


Somatic loss of wild-type alleles can produce disease traits such as neoplasia. Conversely, somatic loss of disease-causing mutations can revert phenotypes; however, these events are infrequently observed. Here we show that ichthyosis with confetti, a severe, sporadic skin disease in humans, is associated with thousands of revertant clones of normal skin that arise from loss of heterozygosity on chromosome 17q via mitotic recombination. This allowed us to map and identify disease-causing mutations in the gene encoding keratin 10 (KRT10); all result in frameshifts into the same alternative reading frame, producing an arginine-rich C-terminal peptide that redirects keratin 10 from the cytokeratin filament network to the nucleolus. The high frequency of somatic reversion in ichthyosis with confetti suggests that revertant stem cell clones are under strong positive selection and/or that the rate of mitotic recombination is elevated in individuals with this disorder.

Ichthyosis with confetti (IWC; also known as ichtyose en confettis, congenital reticular ichthyosiform erythroderma, and ichthyosis variegata) is a very rare, sporadic severe skin disease of unknown cause (13). Affected subjects are born with erythroderma (red skin) owing to defective skin barrier function, prominent scale, and palmoplantar keratoderma (thickening of skin on palms and soles). Poor skin integrity leads to bacterial infections and, frequently, impaired growth and development. Early in life, hundreds to thousands of pale confetti-like spots appear across the body surface and increase in number and size with time (Fig. 1, A and B). Histology of ichthyotic skin shows epidermal thickening and disordered differentiation above the basal layer, with perinuclear vacuolization, lack of a granular layer, and hyperkeratosis (thickening) with retained nuclei in the stratum corneum (Fig. 1, C and D).

Fig. 1

Frequent revertants in ichthyosis with confetti. (A and B) The backs of an 18-year-old female subject (103-1) and 42-year-old male (104-1) show background redness and scaling with hundreds of white, normal-appearing “confetti” spots. (C) Histology of normal human skin showing basal layer (labeled B), stratum spinosum (S), granular layer (G), and stratum corneum(SC). (D) Affected skin shows loss of differentiation of all layers above the basal layer and hypercellularity with increased epidermal thickness. There is no granular layer, and marked perinuclear vacuolization in the suprabasal epidermis and retention of cell nuclei in the stratum corneum are seen. (E) Revertant skin shows normalization of epidermal thickness and architecture, with normal granular layer, normal spinous layer, and stratum corneum. Scale bars in (C) to (E), 50 μm. (F) High-power view of spinous layer in normal epidermis with intercellular spines visible, overlying granular layer with purple keratohyalin granules and basket weave stratum corneum. (G) High-power view of affected skin shows perinuclear vacuolization (black arrows), lack of keratohyalin granules, and retained nuclei (white arrows) in the stratum corneum. (H) High-power view of revertant skin shows normal spinous layer with intercellular spines, granular layer with purple keratohyalin granules in keratinocytes, and basket-weave stratum corneum. Scale bars in (F) to (H), 25 μm.

We studied seven kindreds with characteristic IWC (fig. S1). In five kindreds, there was a single affected offspring of unaffected, unrelated parents, and in two an affected parent had affected offspring. Biopsy of confetti spots in different kindreds revealed that these have normal histology (Fig. 1, E to H), consistent with each representing a revertant from clonal expansion of a normal stem cell. This observation suggested that IWC might be caused by dominant mutations that are lost in revertant spots, and the high frequency of reversion suggested deletion, gene conversion, or recombination as possible mechanisms. To test this, we compared genotypes of DNA from blood and cultured keratinocytes from biopsies of diseased and revertant skin of subject 106-1 typed on Illumina arrays (4). In contrast to blood and disease keratinocytes, revertant DNA showed a single large segment of copy-neutral loss of heterozygosity (LOH) on chromosome 17q extending from 34.5 megabase (Mb) to the telomere at 78.7 Mb (Fig. 2A and fig. S2). Three additional revertant spots from this subject also showed copy-neutral LOH extending from proximal 17q to the telomere, each with different inferred start sites for LOH (Fig. 2B), which excludes simple genetic mosaicism. These findings are consistent with mitotic recombination as the mechanism of LOH (Fig. 2C). In each revertant, the same parental haplotype was lost, consistent with loss of a dominant mutation. We then analyzed 28 revertant spots from five additional patients. Again, all revertants showed copy-neutral LOH on 17q extending to the telomere (Fig. 2B). Sites of inferred recombination are distinct and are confined to the interval from 21.7 Mb (near the centromere) to 34.5 Mb.

Fig. 2

Revertant spots show loss of heterozygosity on 17q. (A) Genotypes on chromosome 17 from revertant keratinocytes of IWC subject 106-1 are shown. From 17pter to 34.5 Mb, genotypes show the expected heterozygosity with genotypes identical to blood and disease keratinocyte DNA (fig. S2), whereas from 34.5 Mb to 17qter, genotypes are homozygous with no change in diploid copy number (fig. S2). (B) Results of genotyping keratinocytes of 32 revertant spots from seven unrelated IWC subjects (106-1 revertants denoted with an asterisk). Gray lines, genomic segments with heterozygous genotypes matching blood DNA; blue lines, segments showing copy-neutral LOH. These results are consistent with the hypothesis that IWCs are caused by a dominant allele distal to 34.5 Mb that is lost by mitotic recombination, as depicted in (C).

These observations suggest that IWC is genetically homogeneous and localize the disease locus to a 99.9% confidence interval, calculated from the position of the most distal recombinant and the number of independent recombinants, to the 34.5 to 37.7 Mb interval on 17q. This interval is notable for a gene cluster encoding 28 type-1 keratins and 24 keratin-associated proteins (5).

Assuming that affected offspring of unaffected parents harbor de novo mutations in the IWC gene, we conducted Illumina sequencing of overlapping polymerase chain reaction (PCR) amplicons spanning the entire critical interval in a parent-offspring trio. At mean 95× per base coverage, ~95% of all the bases in the 99.9% confidence interval were read at least 10 times in each subject, which enabled high-quality genotype calls. The affected subject had a single de novo mutation (table S1), which was confirmed by Sanger sequencing (Fig. 3A). The mutation abolishes the canonical splice acceptor site of intron 6 of keratin 10 (KRT10) (TAG to TGG mutation). Moreover, the mutation was absent in revertant spots (fig. S3). Sequencing of KRT10 transcripts from mutant keratinocyte cDNA revealed a wild-type and a mutant isoform showing splicing at an AG site at bases 7 to 8 in the normal exon 7, leading to an eight-base deletion (Fig. 3, B and C). This results in a frameshift at normal codon 458, leading to 119 aberrant amino acids followed by termination at codon 577. The frameshift peptide has an extremely skewed amino acid composition, with 67 arginine residues (Fig. 3D).

Fig. 3

Mutations in KRT10 (keratin 10) are associated with IWC. (A) Sanger sequencing confirms de novo mutation in KRT10 from Illumina sequencing that is absent in the parents (107-2 and 107-3) but present in the affected offspring (107-1) that abolishes the splice acceptor site of intron 6. (B) Abnormal splicing of KRT10. cDNA from diseased keratinocytes of 107-1 shows two splice forms, one wild-type (WT) and one using an AG splice acceptor that deletes eight bases from WT cDNA (underlined). (C) The genomic structure of KRT10 is shown. The locations of mutations found in IWC kindreds are indicated. (D) IWC frameshifts all produce an arginine-rich C-terminal peptide. The normal sequence of the C-terminal 226 amino acids of keratin 10 is shown; below, in red, the sequence of the frameshift peptides found in IWC are shown, with the position of the frameshift in each kindred indicated.

Sequencing of KRT10 from genomic DNA and disease keratinocyte cDNA in the six other IWC kindreds identified de novo mutations in all four simplex kindreds and transmitted mutations in the two multiplex kindreds (table S2; Fig. 3, C and D; and fig. S4). It is noteworthy that all mutations resulted in cDNAs encoding frameshifts that enter the same alternative C-terminal reading frame (Fig. 3, C and D, and fig. S5). Mutations included two additional intron 6 splice acceptor mutations, an intron 6 splice donor site mutation that results in skipping of exon 6, two frameshift mutations in exon 7, and an exon 6 mutation that creates a premature splice donor site. All of these mutations are absent among control chromosomes, and each is lost in revertant spots (fig. S3). On the basis of these findings, we conclude that mutations in KRT10 cause IWC.

K10 is highly expressed in the suprabasal layers of the epidermis and forms heterodimers with keratin 1 (6, 7), which then assemble to form 10-nm intermediate filaments (8). In diseased skin, keratin 10 levels are reduced (fig. S6, A and B), and electron microscopy reveals a marked reduction in the total number of cytokeratin filaments and poor investment of desmosomes with filaments (fig. S7). In addition, however, K10 is also mislocalized in diseased skin, with prominent nuclear localization in discrete foci that prove to be nucleoli when costained with fibrillarin (Fig. 4, A to F); this nuclear localization is not seen in normal or revertant skin. Keratin 1 shows similar mislocalization in diseased skin (fig. S6, C to E). Similarly, although wild-type and C-terminal–truncated K10 localize to the cytoplasmic filament network when expressed in PLC cells, K10 harboring disease-causing frameshifts localizes exclusively to the nucleolus (Fig. 4, G to L).

Fig. 4

Mutant keratin 10 is redirected to nucleoli in vivo and in vitro. (A to C) Images of normal, mutant, and revertant skin stained with 4′,6′-diamidino-2-phenylindole (DAPI) and antibodies to keratin 10 reveal discrete foci of nuclear keratin 10 in mutant skin which are absent in normal and revertant skin. Scale bars, 50 μm. (D to F) Costaining with the nucleolar marker fibrillarin shows that keratin 10 is in the nucleolus. Scale bars, 10 μm. (G to I) Constructs bearing wild-type keratin 10 (G), keratin 10 truncated at the beginning of the tail domain (amino acid 459) (H), and keratin 10 with the kindred 106 frameshift mutation beginning at codon 460 (I) were expressed in human hepatoma PLC cells and stained with DAPI and monoclonal antibody to keratin 10. Wild-type and “tailless” K10 integrate into the cytoplasmic filament network, whereas the IWC mutant K10 localized to nucleoli as shown by costaining with fibrillarin (J to L). Scale bars (J) to (L), 10 μm.

In summary, we demonstrate that IWC is associated with dominant mutations in keratin 10, all of which produce an arginine-rich C-terminal peptide that causes mislocalization of the protein to the nucleolus. This mislocalization provides a mechanism for disruption of the keratin filament network, which in turn contributes to loss of barrier function. The observed abnormalities in differentiation are unlikely to be due simply to loss of the keratin network, and we speculate that the mutant K10 may disrupt cellular physiology in additional ways, perhaps through effects on ribosomal biogenesis or cell cycle regulation, a process to which K10 has been linked (9). We hypothesize that the nucleolar localization of mutant K10 may be due to RNA binding, owing to the extremely arginine-rich frameshift peptide and the high concentration of ribosomal RNA in the ribosome assembly factory. Most RNA-binding proteins have arginine-rich motifs that interact with the phosphate backbone of RNA, and the specific arginine-rich sequences capable of binding to RNA are diverse (10, 11). Similarly, arginine-rich motifs also contribute to nuclear localization (12). Although we have not seen localization of the frameshift peptide to other RNA- or DNA-containing structures, we cannot exclude this possibility.

IWC is remarkable for its high frequency of spontaneous reversion, with more than a thousand revertants in many subjects. The mechanism—mitotic recombination—represents the complement of a mechanism for producing somatic homozygosity for tumor suppressor mutations (1315). The revertants in IWC are clonal, detectable in the first year of life, and widely distributed in both sun-exposed and unexposed skin. These recombination events simultaneously create homozygous mutant cells, but we see no phenotypic evidence of these, which suggests that they are lethal to cells or contribute little to the epidermal surface.

Somatic reversion has previously been reported for several other disorders (16). These include recessive diseases such as Bloom syndrome and Fanconi anemia and X-linked Wiscott-Aldrich syndrome in which 10 to 20% of patients show some revertant blood cells (1719). These revertant blood cell clones arise by various mechanisms, including intragenic recombination, gene conversion, second-site complementation, and direct reversion. In Bloom syndrome, these revertant clones contributed to refined mapping of the disease locus, analogous to the mapping approach used herein (20).

Similarly, mutations underlying a substantial number of other severe dominant skin diseases have been identified, including keratitis-ichthyosis-deafness syndrome, progressive symmetric erythrokeratoderma, ichthyosis bullosa of Siemens, and dominant dystrophic epidermolysis bullosa; to our knowledge, only a single revertant clone, produced by second-site complementation, has been reported for these diseases (21). Similarly, a fraction of patients with recessive skin disorders have been reported to have revertant patches of skin comprising one to six reported patches in a total of seven patients. In cases where the mechnism was established, all arose by second-site mutation or gene conversion (2227). Moreover, no revertant clones have been reported, or seen in our clinics, in patients with dominant negative or recessive mutations in keratin 10 that cause a distinct disease, epidermolytic ichthyosis (also known as epidermolytic hyperkeratosis) (fig. S8) (28). These exceptions underscore the infrequency of spontaneous reversion and the generally low frequency of mitotic recombination as a mechanism of reversion. In particular, the absence of reversion of other dominant missense mutations in KRT10 implicates the IWC frameshift mutations in the appearance of revertant clones.

The high frequency of somatic reversion in patients with IWC suggests that revertant stem cell clones are under strong positive selection and/or that the rate of production of revertant clones is markedly elevated. The persistence of revertant clones indicates that the reversion event must occur in epidermal stem cells. Epidermal stem cell units have been estimated to populate a fixed area of approximately 0.25 to 0.5 mm2 in human skin (29, 30). The revertant clones we observe in adults with IWC increase in size with time and reach up to 4 cm, consistent with positive selection. Nonetheless, rare revertants in other skin diseases have achieved very large size (2227), from which it may be argued that positive selection is not likely the sole rate-limiting step in production of detectable revertants; however, it suggests an increased rate of mitotic recombination in IWC. Similarly, the fact that none of the previously described revertants in other skin diseases, but all of the IWC revertants, have occurred via mitotic recombination lends support to an effect on the rate of mitotic recombination.

Both mechanisms would be most readily explained by effects of the mutant peptide in epidermal stem cells: Toxic effects could give revertants a survival or replicative advantage; effects on DNA replication, repair, or cell cycle could also promote mitotic recombination. Although K10 is classically regarded as an early differentiation marker, there is evidence that a small proportion of basal cells, which contain rare epidermal stem cells, express KRT10. Moreover, purified putative stem cells of the interfollicular epidermis and follicular bulge show substantial KRT10 expression in proliferating cells, which supports this possibility (3032).

Genetic therapies for dominant diseases have focused on correction of mutations (33) or inhibition of mutant protein synthesis by antisense or interfering RNAs (34). Our results raise the possibility that induction and/or selection of mitotic recombination could be exploited for therapeutic benefit to accomplish cellular reversion of other disease-causing mutations. In this scenario, however, the potential adverse effects of producing homozygosity at undesired loci across the genome would have to be carefully considered in conjunction with the potential benefit of reversion.

Supporting Online Material

Materials and Methods

Figs. S1 to S8

Tables S1 and S2


References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. We thank the patients studied and their families for their invaluable contributions to this study. We thank L. Boyden and S. Baserga for helpful discussions; N. DeSilva, S. Mane, and the Yale Center for Genome Analysis for assistance in development of critical methodologies; and M. Ceneri for the identification of a previously unreported IWC kindred. The National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), NIH, and the Yale Skin Diseases Research Center provided cell culture support for this work. K.A.C. is supported by a K08 award from NIAMS and a fellowship from the Foundation for Ichthyosis and Related Skin Types. Supported in part by a National Center for Research Resources High-End Instrumentation Grant, the Yale Clinical and Translational Science Award, and the Yale Center for Human Genetics and Genomics. R.P.L. is an investigator of the Howard Hughes Medical Institute.
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