Monoallelic Mutations in the Translation Initiation Codon of KLHL24 Cause Skin Fragility Yinghong He, Kristin Maier, Juna Leppert, Ingrid Hausser, Agnes.

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KLHL24: Generalized epidermolysis bullosa simplex (skin fragility)

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Monoallelic Mutations in the Translation Initiation Codon of KLHL24 Cause Skin Fragility Yinghong He, Kristin Maier, Juna Leppert, Ingrid Hausser, Agnes Schwieger-Briel, Lisa Weibel, Martin Theiler, Dimitra Kiritsi, Hauke Busch, Melanie Boerries, Katariina Hannula-Jouppi, Hannele Heikkilä, Kaisa Tasanen, Daniele Castiglia, Giovanna Zambruno, Cristina Has The American Journal of Human Genetics Volume 99, Issue 6, Pages 1395-1404 (December 2016) DOI: 10.1016/j.ajhg.2016.11.005 Copyright © 2016 American Society of Human Genetics Terms and Conditions

Figure 1 Clinical Features and Morphological Examinations of the Skin of Individuals with KLHL24 Mutations (A) Pictures of individual BIII.1 soon after birth and at 1 and 6 years of age demonstrate congenital skin defects, hypopigmentation with a whirled pattern, atrophy and linear scars on the dorsal aspects of the hands and on the lower legs, and thickened nails. Arrowheads point to the clinical features mentioned above. (B) Pictures of individual BII.1 show sparse scalp hair, skin atrophy, palmoplantar keratoderma, and onychogryphosis of the toenails. (C) Pedigree of family B. Dashes indicate that the mutation was excluded. (D) H&E staining of skin sections obtained from the unblistered skin of BII.1 after rubbing demonstrates cleavage at the level of basal keratinocytes, which seem to lyse and loose adhesion (arrowheads). Scale bar represents 50 μm. (E) TEM micrographs of skin samples of CII.2. The upper panels demonstrate cleavage deep in the basal epidermal keratinocytes, just above the hemidesmosomes. Lamina densa of the basement membrane is indicated by arrows. The right panel is a 4-fold magnification of the marked area. The lower panel shows keratin IFs (arrows). (F) Immunofluorescence staining of plectin and integrin β4 in control skin (Co) and in skin sections obtained from EII.1. Note the presence of the markers at the blister base. Blisters are indicated by asterisks. Scale bar represents 50 μm. The American Journal of Human Genetics 2016 99, 1395-1404DOI: (10.1016/j.ajhg.2016.11.005) Copyright © 2016 American Society of Human Genetics Terms and Conditions

Figure 2 Mutations Affecting the Translation Initiation Codon of KLHL24 and Their Consequences (A and B) Sanger sequencing confirmed the KLHL24 mutations and their segregation with the phenotype. Partial KLHL24 sequences of members of families B (A) and E (B) are shown as examples. Note that in both families, the mutation occurred de novo. (C) Schematic representation of the predicted usage of a downstream translation initiation codon due to the mutations. (D) Recombinant expression of GFP-fused normal (KL-GFP) and mutant (KL_1A>G-GFP and KL_2T>C-GFP) full-length and short (cDNA sequence ending in exon 4) KLHL24 polypeptides detected with antibodies to GFP demonstrates slightly faster migration of the mutants. The molecular weight is indicated on the left side in kDa. The pictures represent three technical and three biological replicates. (E) RT-PCR with mRNA from keratinocytes of DIII.1 and EII.1 and normal human keratinocytes from two different donors (NHK and NHK-1) shows that KLHL24 mRNA expression and splicing are not affected by the mutations of the start codon. RT-PCR with primers to GAPDH was run to control loading. Pictures represent experiments done with three biological replicates. MWM indicates the molecular-weight marker in base pairs. (F) Distribution of KLHL24 in the cytoplasm and at the periphery of normal human keratinocytes (NHK) and of keratinocytes treated with scramble siRNA. Staining intensity is reduced in keratinocytes treated with KLHL24 specific siRNA and in DIII.1. Pictures represent three independent experiments. Scale bars represent 20 μm. (G and H) Immunostaining of keratins 5, 14, and 15 in skin samples (G) and in keratinocytes (H) from a normal control individual (Co and NHK) and from DIII.1. Inset in the middle lower panel of (H) is a 2-fold magnification of the marked area. Similar results were obtained with samples from the other individuals. Scale bars represent 50 μm (G) and 20 μm (H). (I) Immunoblotting of whole-cell lysates with antibodies to keratins 5, 14, and 15. GAPDH antibodies were used to control loading. The American Journal of Human Genetics 2016 99, 1395-1404DOI: (10.1016/j.ajhg.2016.11.005) Copyright © 2016 American Society of Human Genetics Terms and Conditions

Figure 3 Alterations of Keratin 14 and Ubiquitin in KLHL24 Mutant Cells (A) Keratin 14 staining was disturbed in keratinocytes of DIII.1. After heat stress, remodeling (perinuclear distribution) and fragmentation of the keratin IFs was observed in the keratinocytes of DIII.1. Similar results were obtained with keratinocytes of EII.1. Scale bar represents 20 μm. (B) Quantification of the percentage of cells (mean values ± SEM) with disturbed keratin 14 staining (10.10% ± 1.05% of 270 NHK cells, 42.74% ± 3.43% of 491 DIII.1 cells, 13.93% ± 1.86% of 263 NHK + heat cells, and 84.8% ± 5.01% of 407 DIII.1 + heat cells; ∗∗∗p < 0.001). All data are from independent biological replicates, and statistical analysis was performed with an unpaired t test with Welch’s correction. (C) Immunoblots with lysates obtained from NHK and keratinocytes of DIII.1, with and without heat stress, were detected with antibodies to ubiquitin and GAPDH. (D) Confocal z stack images of the keratinocytes stained with keratin 14 and ubiquitin antibodies demonstrate colocalization of these markers in the cytoplasm of normal human keratinocytes or keratinocytes of a person with EB simplex due to the keratin 14 variant p.Arg125Cys (EBS-K14R125C). In the keratinocytes of DIII.1, ubiquitin did not demonstrate any cytoplasmic localization. Scale bar represents 20 μm. (E) HaCat cells were transfected with the normal (KL-GFP) and mutant KLHL24 cDNAs (KL_1A>G-GFP and KL_ 2T>C-GFP) and stained with antibodies to keratin 14 (red) and GFP (green). Scale bars represent 20 μm. (F) The graph represents the percentage of keratin-14-positive cells (mean values ± SEM) among those expressing recombinant normal (KL-GFP) or mutant KLHL24 cDNAs (27.66% ± 5.48% of 166 KL-GFP cells, 58.94% ± 5.89% of 120 KL_1A>G-GFP cells, and 51.12% ± 5.63% of 147 KL_2T>C-GFP cells; ∗∗∗p < 0.001 and ∗∗p < 0.01). Data are from three independent experiments. Statistical analysis was performed with an unpaired t test with Welch’s correction. The American Journal of Human Genetics 2016 99, 1395-1404DOI: (10.1016/j.ajhg.2016.11.005) Copyright © 2016 American Society of Human Genetics Terms and Conditions