Whole exome sequencing in Alopecia Areata identifies rare variants in KRT82

Abstract

Alopecia areata is a complex genetic disease that results in hair loss due to the autoimmune-mediated attack of the hair follicle. We previously defined a role for both rare and common variants in our earlier GWAS and linkage studies. Here, we identify rare variants contributing to Alopecia Areata using a whole exome sequencing and gene-level burden analyses approach on 849 Alopecia Areata patients compared to 15,640 controls. KRT82 is identified as an Alopecia Areata risk gene with rare damaging variants in 51 heterozygous Alopecia Areata individuals (6.01%), achieving genome-wide significance (p = 2.18E-07). KRT82 encodes a hair-specific type II keratin that is exclusively expressed in the hair shaft cuticle during anagen phase, and its expression is decreased in Alopecia Areata patient skin and hair follicles. Finally, we find that cases with an identified damaging KRT82 variant and reduced KRT82 expression have elevated perifollicular CD8 infiltrates. In this work, we utilize whole exome sequencing to successfully identify a significant Alopecia Areata disease-relevant gene, KRT82, and reveal a proposed mechanism for rare variant predisposition leading to disrupted hair shaft integrity.

Fig. 1. Gene-level collapsing models identify significant rare variation in KRT82.

Q–Q plots of a Loss of Function (LOF) Model. b LOF + damaging missense Model. c LOF + missense + splicing variants predicted by high TraP scores (≥0.2). The genomic inflation factor (λ) is 1.03, 1.18, 1.23, respectively. The blue line represents the null hypothesis (observed p-values correspond to expected p-values), the yellow line marks the 2.5th percentile of expected p-values, and the green line marks the 97.5th percentile of expected p-values. p-value was determined by two-tailed Fisher’s exact test (FET).

Fig. 2. KRT82 variants are localized to highly conserved disease-annotated domains.

a Table of 9 of the KRT82 damaging variants that fall in a domain previously associated with pathogenic keratin variants in other diseases. b schematic representation of KRT82 with domains (turquoise), AA variants (blue), and conservation score (purple) (1 = highly conserved). c Conservation of KRT82 amino acids across 7 different species: human, mouse, sheep, rhesus monkey, sea lion, vampire bat, and armadillo. AA variants are highlighted in red (LoF), blue (Missense), and green (Splicing). Amino acid change of variant is annotated in red above. EBS = epidermolysis bullosa simplex; CC = Cryptogenic Cirrhosis; DNEPPK = diffuse non-epidermolytic palmoplantar keratoderma; BCIE = bullous congenital ichthyosiform erythroderma; IBS = ichthyosis bullosa of Siemens; MECD = Meesmann’s epithelial corneal dystrophy; WSN = white sponge nevus; PC = pachyonychia congenita; FNEPPK = focal non-epidermolytic palmoplantar keratoderma; SM = steatocystoma multiplex; EPPK = epidermolytic palmoplantar keratoderma; M = monilethrix.

Fig. 3. Patients with KRT82 variants confirmed to have AA.

a Clinical photos of two patients with a missense and nonsense (LOF) variant in KRT82, respectively. Both show hair loss patterns characteristic of AA. b Immunohistochemistry (IHC) staining of CD8+ T cells in 4 patients with missense variants in KRT82. Perifollicular CD8+ T cell infiltrate is a diagnostic characteristic of AA. Each biopsy shown is taken from an individual with AA, serving as 4 biological replicates. c RNA expression profiles of the ALADIN (Alopecia Areata Disease Severity Index) inflammatory signature confirms that three patients (green bars) with KRT82 variants (splice and missense) have increased expression of the inflammatory signature, characteristic of AA. Additionally, these three KRT82-carrying patients (green) cluster within AA patients (AAP-red, ATAU-orange), and not with controls (blue).

Fig. 4. Type II keratin genes cluster together on Ch12.

700 kb chromosomal locus encompassing the type II keratin gene cluster (green bars) on chromosome 12. KRT82 is boxed, in red.

Fig. 5. Haplotype analysis identifies Arg47X as a hotspot mutation.

a 15 AA patients heterozygous for the Arg47X variant (12-52799923-G-A; highlighted in pink). Different genotypes (blue = alternate homozygous, yellow = heterozygous, green = reference homozygous) at variant 12-52799341-C-T (582 bp away) confirms the presence of the Arg47X A allele on different haplotypes, identifying it as a hotspot mutation. b Schematic of KRT82 exon 1 (blue) with ENCODE methylation patterns (orange) in 4 skin cell types: fibroblasts (Fibrobl), Melanocytes (Melano), Neonatal Dermal Fibroblasts (NHDF-neo), and Skin Tissue (Skin BC). This is a visualization of the methylated cytosine (C) of the sense strand that is spontaneously deaminated resulting in a mutational hotspot and premature stop codon at position 47.

Fig. 6. KRT82 is expressed exclusively in anagen.

a IF of KRT82 (green) in postnatal (p) mouse skin. Mouse HFs are in telogen at postnatal days 22 and 90 (p22, p90), in anagen at p31, and catagen at p43. Cuticular staining of KRT82 is only observed in the p31 (anagen) HF. White dashed line outlines the hair shaft cuticle. Weak staining observed in mouse epidermis may be a result of green autofluorescent properties of skin epidermal cells. IF staining was repeated at 3 other anagen timepoints and 2 additional telogen timepoints with similar results. b Western blot analysis of whole mouse skin at varying stages of the hair cycle shows that keratin protein is only present in the anagen phase (p31) of the mouse hair cycle. Western blot was repeated with similar results 2 additional times. Source data are provided as a Source data file.

Fig. 7. Functional KRT82 expression is reduced in AA HF and skin.

a IF staining of KRT82 (green) in human healthy control and AA HF. Dashed line outlines the cuticle, dotted line represents the Line of Auber. IF staining with similar results was repeated in 1 additional control sample and 2 additional AA samples (biological replicates) b RNA expression of KRT82 from whole skin scalp biopsies of control, AAP nonlesional sites, AAP lesional sites, and AT/AU sites. Each circle represents one sample. Statistical analysis was performed using two-tailed unpaired t-test with Welch’s correction. *p = 0.028, ****p < 0.0001. n = 98 individuals (36 controls, 30 AAP, 32 ATAU). c Scalp RNA expression of patients carrying KRT82 variants at nonlesional (Variant Nonlesional) and lesional (Variant Lesional) sites compared to AA patients without KRT82 variants (Nonlesional and Lesional) and controls without AA or KRT82 variants. n = 32 individuals (12 controls and 20 AAP patients). Expression data is displayed in boxplots in which each sample data point is represented by individual dots; the center line denotes the median; upper and lower limits of the box denote 75th and 25th percentiles, respectively; and the whiskers indicate the dataset maximum and minimum. d IHC of KRT82 and CD8 in healthy control and lesional biopsies in three AA patients with missense variants. AA biopsies are from individual patients and serve as 3 biological replicates. Staining in another control biopsy was not repeated; however, images were taken of other hair follicles, serving as technical replicates.