IDENTIFICATION OF CANDIDATE VARIANTS IN PATIENTS WITH CCCA
Figure 1.
Clinical Features and Mutation Analysis in Central Centrifugal Cicatricial Alopecia (CCCA).
We initially conducted exome sequencing in a discovery set, which included 16 women of African ancestry who had received a diagnosis of CCCA (Table S2 in the Supplementary Appendix). The discovery set included one familial case (from Family 1) (Fig. S1 in the Supplementary Appendix). Each patient received a clinical diagnosis from a dermatologist at each respective location. For each patient, the diagnosis was confirmed by means of biopsy (Figure 1). Clinical examination revealed hair loss over the crown, with centrifugal spread and a perifollicular grayish halo on dermoscopy in all patients. All the biopsy specimens showed decreased hair-follicle density and perifollicular lymphocytic infiltration with areas of fibrosis. CCCA was graded in all the patients according to the Central Hair Loss Grading scale (scores range from 0 to 5, with higher scores indicating more severe disease). Patients who were included in the discovery set had a moderate-to-severe condition (score of 3 to 5) (Table S2 in the Supplementary Appendix).10
Exome sequencing was performed by Fulgent Genetics in Patients 1 through 10 and by BGI in Patients 11 through 16. Variants that were identified by means of exome sequencing were classified according to their predicted effects on protein function with the use of the PolyPhen-2 (Polymorphism Phenotyping, version 2) tool,11 Provean (Protein Variation Effect Analyzer) software,12 the SIFT (Sorting Intolerant from Tolerant) algorithm,13 and the ConSurf server.14 The prevalence of CCCA is estimated to be 2.7% among black women in South Africa5 and 5.6% among black women in the United States.6 Because CCCA has been reported almost exclusively in women of African ancestry,4,15,16 we selected for further analysis variants that were predicted to be pathogenic, that were shared by the patients, and that had a minor allele frequency of less than 0.05 in the African population and of less than 10−4 in the European population. (Prevalence data were derived from the Genome Aggregation Database [http://gnomad.broadinstitute.org/. opens in new tab].)
Table 1.
Variants Identified in PADI3 in Patients with Central Centrifugal Cicatricial Alopecia.
Using this strategy, we identified four heterozygous mutations in the gene PADI3 (RefSeq accession number, NM_016233.2. opens in new tab) in 5 of 16 patients (31%): c.856A→G, c.1744G→A, c.1669C→T, and c.832-2A→G (Figure 1C and Table 1). PADI3 encodes the enzyme peptidyl arginine deiminase, type III. These four mutations included one splice-site mutation and three missense mutations. All the missense mutations had a minor allele frequency in the range of 0.0001 to 0.04 in the African population while being very rare among persons of European ancestry (Table 1). The missense mutations are predicted to have a deleterious effect on protein function (Table S3 in the Supplementary Appendix). The mutant amino acids are located in the second immunoglobulin-like domain or the catalytic domain of the enzyme (Figure 1D). Protein modeling suggested that these mutations would be likely to result in protein misfolding (Fig. S2 in the Supplementary Appendix).17,18The splice-site mutation c.832-2A→G is expected by several prediction tools19-22 to abrogate the acceptor splice site of intron 7 and to effect skipping of exon 8, which in turn is expected to lead to a frame shift.
CONSEQUENCES OF CCCA-ASSOCIATED MUTATIONS IN PADI3
PADI317 is a member of the peptidyl arginine deiminase family of enzymes, which are responsible for catalyzing the post-translational deimination of proteins by converting positively charged l-arginine residues into citrullines in the presence of calcium ions.23 They have distinct substrate specificities and tissue-specific expression patterns.23,24 PADI3 is detected mainly in the epidermis and hair follicles.25,26 In the skin, it is responsible for mediating the modification of proteins critical for normal hair-shaft formation and shaping, such as trichohyalin, and may also play a role in interfollicular epidermal differentiation.23
Although PADI3 has been associated with abnormal hair formation in patients who have the uncombable hair syndrome (Online Mendelian Inheritance in Man number, 191480. opens in new tab),9 it has been unclear whether it has a role in the pathogenesis of CCCA. In an attempt to obtain further in vivo evidence of the relevance of CCCA-associated mutations to the disease manifestations, we used deep sequencing of RNA extracted from biopsy samples of scalp skin obtained from three patients with CCCA who had mutations in PADI3 and from four healthy controls who were matched for ancestry population, age, and sex.
The expression of numerous genes differed between scalp-skin samples obtained from patients with CCCA and control samples (Fig. S3A and Table S4 in the Supplementary Appendix). Expression of PADI3was markedly lower in the skin of patients with CCCA than in the skin of controls (Table S4 in the Supplementary Appendix), as was the expression of genes encoding several peptidyl arginine deiminase substrates (including TCHH9 and S100A327), those known to be related to hair loss (including LIPH,28DSG4,29 HR,30 and CDSN31), and those encoding hair keratins and keratin-associated proteins (which contribute to the normal structure of hair fibers32). Ingenuity pathway analysis revealed that the expression of many genes encoding molecules that play a central role in hair-follicle development was reduced overall in the skin of patients with CCCA. Relevant RNA-sequencing data were validated with the use of quantitative reverse-transcriptase polymerase chain reaction. Details are provided in Figures S3 through S5 in the Supplementary Appendix.
Figure 2.
Consequences of CCCA-Associated Variants inPADI3.
To further investigate the consequences of the CCCA-associated missense mutations in PADI3, HaCaT (a human keratinocyte cell line) cells were transiently transfected with constructs encoding nonvariant and mutated PADI3. Immunoblotting of cell extracts showed expression of all three mutant PADI3 constructs that was slightly lower than that of the nonvariant construct (Figure 2A). Accordingly, PADI3 expression was reduced in a scalp-skin sample obtained from a patient with CCCA (Figure 2B). We then examined the effect of PADI3mutations on the subcellular location of the enzyme. Immunofluorescence analyses showed a homogeneous cytoplasmic distribution of PADI3 in cells transfected with nonmutated PADI3, as previously shown,9 in contrast with cells transfected (one at a time) with the three mutated PADI3 variants. In these cells, we observed abnormal intracellular localization of the protein with formation of aggregates in the cytoplasm (Figure 2C).
We then assayed enzymatic activity associated with the three mutant constructs, as compared with nonvariant PADI3. A construct with a mutation that had been previously associated with the uncombable hair syndrome33 served as a positive control. We observed a significant decrease in enzymatic activity on transfection of the four constructs into HaCaT cells, as compared with the HaCaT cells transfected with the construct containing nonmutated PADI3 (Figure 2D).
FREQUENCY OF PADI3 MUTATIONS IN CCCA
We then sequenced PADI3 in a replication set, which included 42 patients (Table S2 in the Supplementary Appendix); we observed a PADI3 variant in 9 of them. Altogether, we identified a total of six different mutations in PADI3 (Figure 1C and Table 1), which were present in 14 of the 58 patients (24%) with CCCA who participated in this study. The two familial cases were identified; these patients were members of families with cosegregation of the mutations and affected status (Fig. S1 in the Supplementary Appendix).
In a post hoc analysis, the PADI3 mutation frequency among 58 women of African ancestry who had CCCA (116 alleles) was found to differ significantly from that calculated for a control cohort of women of African ancestry (from the gnomAD V2.1 control set) according to the chi-square test (P=0.002) and Fisher’s exact test (P=0.006). The difference remained significant after adjustment for relatedness of persons according to the chi-square test (P=0.03) and Fisher’s exact test (P=0.04). We did not control for population stratification. However, the mutation frequency was similar across various African subpopulations (Table S5 in the Supplementary Appendix).
