Autosomal-dominant woolly hair resulting from disruption of keratin 74 (KRT74), a potential determinant of human hair texture

Abstract

Autosomal-dominant woolly hair (ADWH) is a rare disorder characterized by tightly curled hair. The molecular basis of ADWH has not previously been reported. In this study, we identified a Pakistani family with ADWH. The family showed linkage to chromosome 12q12-q14.1, containing the type II keratin gene cluster. We discovered a heterozygous mutation, p.Asn148Lys, within the helix initiation motif of the keratin 74 (KRT74) gene in all affected family members. KRT74 encodes the inner root sheath (IRS)-specific epithelial (soft) keratin 74. We demonstrate that the mutant K74 protein results in disruption of keratin intermediate filament formation in cultured cells, most likely in a dominant-negative manner. Furthermore, we sequenced the mouse Krt71-74 genes in the dominant Caracul-like 4 (Cal4) allele, which is characterized by a wavy-coat phenotype and maps to the same region of mouse chromosome 15 as the Caracul (Ca) and Reduced coat (Rco) alleles. We identified a heterozygous mutation, p.Glu440Lys, not in Krt74 but in the neighboring gene, Krt71. Krt71 was previously reported to harbor Ca and Rco mutations, as well as a coding SNP that is associated with curly-coated dogs. In this study, we define the ADWH phenotype resulting from a mutation in a hair-follicle-specific epithelial keratin in humans. Our findings not only further underscore the crucial roles of the IRS-specific epithelial keratin genes Krt71-74 in hair disorders but also open the possibility that these genes might function as genetic determinants of normal variation in hair texture across mammalian species.

Figure 1

Fine Mapping of ADWH Phenotype on Chromosome 12q12-q14.1 (A–C) Clinical appearance of ADWH. (D–G) Appearance of the hair shaft in affected individuals with ADWH. The scale bar represents 100 μm. (H) Pedigree and haplotype analysis of a Pakistani family ADWH1. The linked haplotype is indicated in red. Critical recombination events are indicated by black arrowheads.

Figure 2

Identification of a Mutation in the KRT74 Gene (A) Schematic representation of the candidate region harboring the ADWH gene. The candidate region and the type II keratin gene cluster are boxed in yellow and red, respectively, and localization of four IRS-specific keratin genes, KRT71–74, is shown at the bottom. (B) A heterozygous mutation c.444C > G (p.Asn148Lys) in the KRT74 gene in affected individuals from the family ADWH1. (C) Segregation analysis of the mutation c.444C > G in the KRT74 gene. PCR products of 338 bp from the mutant (Mut) allele were digested into 234 bp and 104 bp fragments with the restriction enzyme AcuI, whereas those from the wild-type (Wt) allele were not digested. Affected individuals are indicated in red and show the digested bands of 234 bp and 104 bp from the Mut allele as well as the undigested band of 338 bp from the Wt allele. MWM, molecular weight markers. C, control individual. (D) Multiple-amino-acid sequence alignment of the helix initiation motif (HIM) of human type II keratins. K74 (K6irs4) is indicated in red. The Asn residue at amino acid position 9 in the HIM, which is invariant and completely conserved among all human type II keratins, is highlighted in yellow.

Figure 3

Mutant K74 Protein Disrupts Endogenous KIF Formation in PtK2 Cells, and K74 Colocalizes with LPAR6 in Huxley's Layer of Human Hair Follicles (A–C) Ectopically expressed wild-type (Wt) K74 protein forms a KIF network via heterodimerization with endogenous K18 protein. (D–F) The p.Asn148Lys mutant (Mut) K74 protein causes a collapse of the endogenous KIF network around the nucleus. (G–I) Double immunostainings with guinea pig polyclonal anti-K74 (1:2,000) (G) and rabbit polyclonal anti-P2Y5 (LPAR6; 1:200; MBL International) (H) antibodies show that both K74 and LPAR6 proteins are coexpressed in Huxley's (Hux) layer of the IRS of human hair follicles from a normal control individual (I), whereas only LPAR6 is expressed in Henle's (He) layer of the IRS (I). The right panels are merged images, and counterstaining with DAPI is shown in blue (C, F, and I). Scale bars represent 20 μm (A) and 100 μm (G).