A Small Deletion Hotspot in the Type II Keratin Gene mK6irs1/Krt2-6g on Mouse Chromosome 15, a Candidate for Causing the Wavy Hair of the Caracul (Ca) Mutation

Corresponding author: Hiromichi Yonekawa, The Tokyo Metropolitan Institute of Medical Science (Rinshoken), 3-18-22, Honkomagome Bunkyo-ku, Tokyo 113-8613, Japan., yonekawa{at}rinshoken.or.jp (E-mail)

Communicating editor: N. TAKAHATA

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

A new mutation has arisen in a colony of mice transgenic for human -galactosidase. The mutation is independent of the transgenic insertion, autosomal dominant, and morphologically very similar to the classical wavy coat mutation, caracul (Ca), on chromosome 15. Therefore, we designated this locus the caracul Rinshoken (Ca^Rin). Applying a positional cloning approach, we identified the mK6irs1/Krt2-6g gene as a strong candidate for Ca^Rin because among five Ca alleles examined mutations always occurred in the highly conserved positions of the -helical rod domain (1A and 2B subdomain) of this putative gene product. The most striking finding is that four independently discovered alleles, the three preexistent alleles Ca^J, Ca^9J, Ca^10J, and our allele Ca^Rin, all share one identical amino acid deletion (N 140 del) and the fifth, Ca^medJ, has an amino acid substitution (A 431 D). These findings indicate that a mutation hotspot exists in the Ca locus. Additionally, we describe a Ca mutant allele induced by ENU mutagenesis, which also possesses an amino acid substitution (L 424 W) in the mK6irs1/Krt2-6g gene. The identification of the Ca candidate gene enables us to further define the nature of the genetic pathway required for hair formation and provides an important new candidate that may be implicated in human hair and skin diseases.

THE keratins constitute a group of >40 highly insoluble proteins that serve as the subunits forming intermediate filament polymers in epithelial cells (O'GUIN et al. 1990 ; FUCHS 1995 ). In addition to the epithelial or soft -keratins, this multigene family also contains a smaller subfamily of hard -keratins, which, because of their most common site of occurrence, are generally referred to as hair keratins. Previous protein studies indicated that, independent of the species, the hair keratin family consists of four individual members per subfamily, which were designated hair acidic, type-I keratin (Krt1) and hair basic, type II keratin (Krt2), respectively (HEID et al. 1986 ; LYNCH et al. 1986 ). Keratins are expressed as obligate heterodimers of Krt1/Krt2 pairs in a tissue- and differentiation-specific fashion.

Keratin gene products play an important role in the mechanical support of hair development. Such a role has been confirmed in the context of transgenic mouse models (POWELL and ROGERS 1990 ; MAGIN 1998 ) and through mutations in several keratin genes that have been found to cause a variety of diseases affecting hair development in humans (IRVINE and MCLEAN 1999 ). Over the past several years, hair keratin research has made considerable progress. Thus, the entire sets of human type I and type II hair keratin genes, as well as the patterns of expression of the encoded proteins, have been elucidated (ROGERS et al. 1998 , ROGERS et al. 2000 ; LANGBEIN et al. 1999 , LANGBEIN et al. 2001 ). In particular, the hair follicle has been found to contain both hair keratins and epithelial keratins. The former are found in the hair fiber and the latter in the inner and outer root sheath. One of the epithelial keratins, mK6IRS1/KRT2-6G, the product of the mK6irs1/Krt2-6g gene, is expressed exclusively in the inner root sheath and could therefore play an important role in hair development and/or hair morphology through its influence on hair follicle cell development, although this simplified scheme can elucidate in part either the processes or the mechanisms on the development.

Regarding the development of hair follicle cells, in the early stage the epithelium arose from the ectoderm, a single layer of pluripotent cells that can differentiate to either follicle or epidermis. The critical process depends upon whether the ectoderm can contact a condensate of specialized mesenchyme called the dermal papillae. A mesenchymal signal triggers an ectodermal cell to proliferate and the cells grow downward to form a hair germ. An ectodermal signal leads these epithelial cells in the dermal papillae to differentiate further and to develop into a hair follicle, forming a compartment of stem cells, a sebaceous gland, and a hair shaft surrounded by an outer and inner root sheath. Although the wingless/integrated and Sonic hedgehog (SHH) signaling pathway clearly participate in the developmental processes of both follicle and epidermis, less is known about the former (HARDY 1992 ; FUCHS 2001 ; MILLER 2002 ).

The periods of hair growth are followed by a regression phase (the catagen phase), when the lower part of the follicle undergoes programmed cell death, and a resting phase (the telogen phase), before onset of a new growth phase (the anagen phase). Cyclical growth of hair continues throughout postnatal life, allows the follicle to remodel itself, and occurs randomly in humans but in a synchronized manner in mice (MILLER 2002 ).

Mouse hair keratin genes colocalize with epithelial keratin genes on the distal portion of chromosome 11 (Krt1) and the distal portion of chromosome 15 (Krt2). In both of these regions there are several previously described mutations that cause abnormal hair: on chromosome 11, the mutations rex (Re), recombination-induced mutation 3 (Rim3), and whiskers amiss (wam) and on chromosome 15, caracul (Ca), shaven (Sha), velvet coat (Ve), naked (N), and Hague (Hag) (DUNN 1937 ; DOOLITTLE et al. 1996 ; SATO et al. 1998 ; TAYLOR et al. 2000 ; POIRIER et al. 2002 ).

While constructing human -galactosidase transgenic mice (KASE et al. 1988 ), we isolated a mutant with an abnormal coat hair phenotype. The new mutant is a single autosomal dominant, and the phenotype has a strong resemblance to that caused by the Ca mutation (DUNN 1937 ). The locus was also mapped distal to chromosome 15, which is very close to the Ca locus; therefore, we named the new mutation caracul Rinshoken (Ca^Rin). Using positional cloning, we discovered that a deletion of one amino acid residue, aspartic acid, had occurred in the mK6irs1/Krt2-6g coding sequence, which is therefore a candidate for the Ca mutation. Next, we searched for mutations in five Ca alleles, each of which had been independently discovered in the Jackson Laboratory (JAX alleles), and sought N-ethyl-N-nitrosourea (ENU)-induced wavy coat hair mutants isolated during the RIKEN mutagenesis project. This study resulted in the identification of mutations in the mK6irs1/Krt2-6g coding sequence of four JAX alleles and one ENU-induced allele. Interestingly, three other alleles possess a deletion identical to that found in Ca^Rin, suggesting that a germline mutation hotspot does exist in the Ca locus.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Mice:
The Ca^Rin mutant arose spontaneously in a C57BL/6slc (B6) mouse during the production of human -galactosidase cDNA transgenics. This founder mouse was fixed as a mutant strain and bred in the animal facility at The Tokyo Metropolitan Institute of Medical Science (Rinshoken). Heterozygous Ca^Rin mice were crossed to MSM/Ms (MSM) or JF1/Ms (JF1) strain animals to generate intersubspecific backcross progeny for linkage analysis. The MSM and JF1 are inbred strains established from the Japanese wild mouse (MORIWAKI 1994 ; KIKKAWA et al. 2001 ) and maintained at the National Institute of Genetics. Five mutants with other Ca alleles, B6C3Fe-a/a-Ca^medJ, BALB/cBy-Ca^10J, BALB/cBy-Ca^9J, C3HeB/FeJ-Ca^J/+Hm/+Sl/+, and C57BL/6By-Ca^Sl, and their background strains were purchased from the Jackson Laboratory (JAX) as genomic DNA samples. Two ENU-induced mutant lines, M100573 and M100689, were provided from RIKEN Genome Science Center (GSC). For mutagenesis, ENU was administered to C57BL/6J male mice (G₀). Sequence analyses were performed using G₂ animals with the genetic background of DBA/2J x (DBA/2J x ENU-treated C57BL/6J)F₁. The detailed protocol of ENU mutagenesis is described on the RIKEN GSC website (http://www.gsc.riken.go.jp/Mouse/).

Linkage analysis:
A total of 321 backcross progeny were typed for the wavy coat hair of the Ca phenotype and then genomic DNA was prepared from liver and/or pinna skin and used for linkage analysis. Microsatellite markers were purchased from Research Genetics/Invitrogen (San Diego). Simple sequence length polymorphisms (PCR-SSLP) were analyzed: 1 µl (100 ng) of genomic DNA was amplified in a total volume of 15 µl with final concentrations of 1x Gold buffer II, 1.5 mM MgCl₂, 200 µM dNTPs, 0.25 µM of each primer, and 0.1 units AmpliTaq Gold polymerase (Applied Biosystems, Foster City, CA). Reactions were carried out in the GeneAmp 9700 thermal cycler with a PCR profile of one cycle at 95° for 5 min and 40 cycles of 94° for 30 sec, 55° for 40 sec, and 72° for 60 sec. PCR products were loaded on 4% agarose gels (3% NuServe agarose and 1% agarose). PCR primers for eight Krt2 genes were designed according to the published mRNA sequences (Table 1). These primer pairs amplified a fragment corresponding to the 5'- and 3'-untranslated region (UTR). The chromosomal location of these genes was determined by linkage analysis through key recombinants by single-strand conformation polymorphism (PCR-SSCP). Linkage analysis was performed with the Map Manager QXP (MANLY et al. 2001 ).

Bacterial artificial chromosome analysis:
A mouse CITB bacterial artificial chromosome (BAC) library was screened at Research Genetics/Invitrogen by PCR with 3'-end markers of Krt2 genes. DNA from positive clones was isolated by the alkaline lysis method (SAMBROOK et al. 1989 ) and then analyzed by pulsed field gel electrophoresis. BAC ends were directly sequenced with T7 and SP6 primers. To determine if their BAC-end-derived sequences mapped to mouse chromosome 15, PCR primers for these ends were synthesized (Table 1) and then used to map these PCR products through key recombinants by PCR-SSCP. Details of these BAC-end-derived primer sequences are given in Table 1.

Mutation screening:
To screen for mutations between B6 and Ca^Rin, total RNA was isolated from 5-week-old mouse skin by using TRIzol (Life Technologies/Invitrogen) following the manufacturer's protocol. cDNA was generated with the Omniscript RT kit (QIAGEN, Valencia, CA) using 1 µg of DNase-pretreated total RNA. The entire genomic region of the mK6irs1/Krt2-6g gene was amplified to give overlapping PCR products from five mutant mice with other Ca alleles; two ENU-induced mutant lines, M100573 and M100689; and their background strains (C57BL/6Jslc, C57BL/6J, BALB/cBy, C3HeB/FeJ, and DBA/2J) by long and accurate (LA) PCR (Takara, Otsu, Japan) using the primers corresponding to each exon: exons 1–7, K6irs5'F and R5; exons 6 and 7, K6irsF5 and R14; exons 7–9, K6irsF7 and R9. Sequences of these primers are shown in Table 2. PCR products were gel purified, sequenced using BigDye Terminator cycle sequencing kits, and analyzed on a 3100 genetic analyzer (Applied Biosystems).

RT-PCR and Northern blot hybridization:
Approximately 1 µg of DNase-pretreated total RNA prepared from cDNA obtained from B6 and B6-Ca^Rin skin (5 weeks old) was reverse transcribed using the Omniscript RT kit. The cDNA was amplified for 30 cycles (94° for 30 sec, 60° for 30 sec, 72° for 1 min) using AmpliTaq Gold and a 9700 Thermocycler (Applied Biosystems). The products were subjected to agarose gel electrophoresis. Primers used for detection of K6irs/Krt2-6g-specific transcripts were K6irsF7 and K6irsR4. This product was a 755-bp stretch derived from the 3' coding sequence and part of the 3'-UTR.

Total RNA (20 µg per lane) was loaded onto a 1% agarose-formaldehyde gel and transferred onto Hybond N+ membrane (Amersham, Arlington Heights, IL). The filter was hybridized with a randomly labeled (Amersham) probe in Rapid-hyb buffer (Amersham) at 70° for 12 hr and then washed (2x SSC, 0.1% SDS) at room temperature for 20 min, followed by stringent washing (0.1x SSC, 0.1% SDS) at 65° for 15 min. The K6irs/Krt2-6g probe was the above-mentioned 755-bp fragment. Blots were stripped and hybridized with a mouse Gapdh probe.

Histological analysis:
Dorsal skin was dissected from B6 and B6-Ca^Rin at 5 weeks of age and fixed in 4% paraformaldehyde overnight. After fixation, the tissues were dehydrated, embedded in paraffin, sectioned (6 µm), and stained with hematoxylin and eosin.

We carried out immunohistofluorescence analysis using polyclonal antibody against mK6IRS1/KRT2-6G, at a dilution of 1:3200. The antibody against mK6IRS1/KRT2-6G was kindly provided by Y. Shimomura and M. Ito (Department of Dermatology, Niigata Graduate School of Medicine & Dental Science, Niigata, Japan; AOKI et al. 2001 ). For immunofluorescence, FITC-coupled rabbit IgG (Molecular Probes, Eugene, OR) was used at a dilution of 1:1000.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Description of phenotype:
During transgenesis experiments with human -galactosidase cDNA (KASE et al. 1988 ), we isolated a mutant mouse carrying an abnormal coat hair phenotype. Southern blot analysis of affected individuals revealed that their genome did not contain any human genetic components, suggesting that the mutation occurred independently in the transgenic mouse colony. Animals carrying the new mutant are easily distinguished from their nonaffected littermates, because of the following phenotype: (1) the mutant mice exhibit rough and greasy fur and (2) their hair is wavy and pointed in different directions (Fig 1A), with the wavy appearance prominent between 3 and 6 weeks of age. Thus, there is a strong resemblance to individuals carrying the Ca mutation (DUNN 1937 ). In the affected progeny, whiskers are markedly curved, and coat hair is wavy from the time of first appearance until postweaning age (4 weeks), as is also the case in the original Ca mutant (Fig 1A and Fig E). After 4 weeks, the waviness of the coat hair became much less apparent, and the hair acquired a plush-like morphology (Fig 1, B–D). An ENU-induced mutant, M100689, also shows a similar phenotype of hair coat and whiskers (Fig 1F).

View larger version (119K): In this window In a new window Download PPT slide   Figure 1. Phenotype of hair coat and whiskers. (A) Hair coat at 3 weeks of age in normal (left) and C57BL/6slc-CaRin (right). Wavy hair coat can be seen in this mutant. (B) Hair coat at 12 weeks of age in normal (left) and C57BL/6slc-CaRin/- (right). After 4 weeks, the wavy coat hair phenotype is less apparent, but the hair looks plush like. (C and D) Comparison of hair texture phenotype between normal (C) and CaRin mouse (D) at 12 weeks. Note the disordered hairs, as at 12 weeks, with irregular curls and kinks in hair of the whole body. (E) Ventral view of head (whiskers) of C57BL/6slc mice comparing normal (left) and CaRin (right). (F) Whiskers and hair coat at 8 weeks of age in an ENU-induced mutant, M100689.

All of the F₁ progeny showed the mutant phenotype using at least 100 progeny in reciprocal crosses (data not shown), with the heterozygotes being phenotypically indistinguishable from the homozygotes. We are thus dealing with a single autosomal dominant mutation. The mutated locus was mapped distal to chromosome 15, very close to the Ca locus, and thus we named the new mutation caracul Rinshoken (Ca^Rin).

Histological observation revealed that the dorsal skin of 5-week-old mutant mice was thinner than that of age-matched controls, mainly because of decreased thickness of the adipose layer (Fig 2A and Fig B). At this age, the dorsal skin hair follicles were at the second anagen stage in both the mutant and control mice. In contrast to hair follicles of nonaffected littermates, the follicles of mutants were curved and twisted randomly, thus producing wavy hair (Fig 2B). The extent of curvature was different in each follicle. In severe cases, curved follicles had grown alongside the subcutaneous muscle layer (Fig 2E and Fig F). Moreover, the mutant follicles exhibited abnormal morphology of the inner root sheath (IRS): namely, the IRS lacked uniformity of thickness and, in some follicles, the IRS cells showed abnormal keratinization (Fig 2C and Fig D). In addition to these abnormalities, follicular-derived cysts containing keratin debris were occasionally observed in the dermis. The mutant mouse sebaceous glands were larger than those of control mice and this was particularly marked around the cyst structures (Fig 2G).

View larger version (124K): In this window In a new window Download PPT slide   Figure 2. Dorsal skin sections from control (A) and CaRin (B) mice at 5 weeks. Normal anagen hair follicles (A) have straight hairs with a tightly compacted IRS structure (C), whereas the follicles of the mutant are twisted and/or curved (B). A follicle in B is twisted at least four times (arrowheads) producing waved hair (arrow). Severely curved follicles in E have grown alongside the subcutaneous layer. One of them is sectioned longitudinally (E, solid arrowhead), and the other is cut transversely (E, open arrowhead). Comparison of sebaceous glands (sg) of control (F) and CaRin (G) mice at 5 weeks. A hair follicle-derived cyst is observed in the dermis. Sebaceous glands are enlarged around the cyst structure (arrows). Hematoxylin and eosin staining; ors, outer root sheath; irs, inner root sheath; cu, cuticle; co, cortex; me, medulla. Bar, 100 µm.

Linkage mapping of Ca^Rin:
In small scale intersubspecific backcrossing between B6-Ca^Rin/Ca^Rin and MSM/Ms (backcross a), we first determined that the Ca^Rin locus mapped to the distal region of chromosome 15 (data not shown). We then extended the number of backcross segregants to 321 and subjected all to Ca phenotyping. They were also genotyped by molecular markers flanking the Ca^Rin locus: D15Mit14 and D15Mi77 [D15Mit14/77] as a proximal marker and D15Mit40 as a distal marker (Fig 3A). There were 19 recombination events between D15Mit14/77 and Ca^Rin and 4 between Ca^Rin and D15Mit40. The Ca^Rin locus was therefore located within this 7.5-cM interval. We also generated other intersubspecific backcrosses between B6-Ca^Rin/Ca^Rin and JF1 (backcross b) and examined 297 segregants. Linkage analysis was done with the same molecular markers as above. The order of the markers was identical to that of backcross a, but there was a striking difference regarding the genetic distances between the adjacent markers. Thus, the distance between D15Mit14/77 and Ca^Rin is 6.2 cM in backcross a, whereas it is only 2.2 cM in backcross b. Similarly, the Ca^Rin and D15Mit15/16 interval is 5.8 cM in backcross a, whereas it is only 0.3 cM in backcross b (Fig 3).

View larger version (9K): In this window In a new window Download PPT slide   Figure 3. Linkage map of the region around the CaRin locus on mouse chromosome 15. (A) Markers shown were typed in 321 progeny from the MSM cross: C57BL/6J x (C57BL/6J-CaRin/CaRin x MSM/Ms)F1. (B) Markers shown were typed in 297 progeny from the JF1 cross: C57BL/6J x (C57BL/6J-CaRin/CaRin x JF1/Ms)F1. Map distances between adjacent loci are shown in centimorgans on the left.

Because the 12 Krt2 loci, Krt2-1, -4, -6a, -6b, -6g, -7, -8, -10, -16, -17, -18, and -19, had all been previously mapped to the Ca^Rin region of mouse chromosome 15 (Mouse Genome Informatics: http://www.informatics.jax.org/), we tried to superimpose these Krt2 loci on our maps. For this purpose, we used the sequence-tagged site (STS) markers derived from the 5'- and 3'-UTR sequences of these Krt2 genes to genotype the loci using PCR-SSCP analysis. Four STS markers from the Krt2 genes mentioned above, Krt2-1 (5'), Krt2-6a (3'), Krt2-10 (3'), and Krt2-17 (5'), mapped to the region critical for the Ca^Rin locus, because the polymorphisms were found in each Krt2-STS marker between B6 and MSM. Linkage showed that the Krt2-10 locus mapped proximal to Ca^Rin with one recombination event; the Krt2-17 locus mapped distal to Ca^Rin with one recombination event; and the Krt2-6a mapped at the same position as Ca^Rin in the backcross a progeny (Fig 3A). On the other hand, linkage showed that Krt2-10/6a mapped proximal to Ca^Rin with three recombination events and that Krt2-1/17 mapped to the same position as Ca^Rin in the backcross b progeny (Fig 3B). The Ca^Rin locus, therefore, was located between Krt2-6a and Krt2-17.

Physical mapping of the Ca^Rin locus:
To construct a physical map of the Ca^Rin region, the linkage map was used as a scaffold to assemble a BAC contig. The core of the physical map was Krt2-6a and Krt2-17, the markers closest to Ca^Rin. Five overlapping BACs (310F1, 298P5, 363H8, 304H2, and 51L7) were isolated from BAC libraries using the STS markers derived from these two Krt2 genes and BAC-end sequences (Fig 4). Further characterization of this BAC contig by PCR-based approaches using STS markers derived from the 5'- and 3'-UTR sequences of the Krt2 gene confirmed that this contig contained the Krt2-6b and mK6irs1/Krt2-6g genes as well as Krt2-6a and Krt2-17. This physical map indicated that the gene order of Krt2 is proximal: Krt2-6a, Krt2-6b, mK6irs1/Krt2-6g, Krt2-17 (Fig 4). Thus, we narrowed down the Ca^Rin nonrecombinant interval and concluded that it contains only four Krt2 genes, namely, Krt2-6a, Krt2-6b, mK6irs1/Krt2-6g, and Krt2-17, which are therefore strong candidates for Ca^Rin.

View larger version (17K): In this window In a new window Download PPT slide   Figure 4. BAC-based physical map. Chromosome 15 is indicated as a horizontal line with the chromosome centromere designated by a solid circle. Krt2 genes are shown as solid boxes overlying the horizontal line. BAC clones are positioned as shaded boxes over the horizontal line and drawn to scale. The marker content of each clone is designated by vertical lines connecting the clone with chromosome 15. The nonrecombinant intervals defined by linkage analysis using two-intersubspecific backcross are also shown. The critical interval is indicated by an arrow.

mK6irs1/Krt2-6g mutations in Ca:
To evaluate these four positional candidates, Krt2-6a, Krt2-6b, mK6irs1/Krt2-6g, and Krt2-17, we carried out RT-PCR analysis to compare the sequences of Ca^Rin and +/+ (B6) skin cDNA. We did not find any mutations in the coding regions of Krt2-6a, Krt2-6b, and Krt2-17 (data not shown). However, in the sequence of the mK6irs1/Krt2-6g gene, we identified a 3-bp deletion (CAA) at nucleotide positions 418–420, causing an asparagine deletion at amino acid position 140, which lies in the -helical rod domain (Fig 5 and Fig 6). Asparagine 140 is highly conserved among other epithelial keratin genes in mouse and human, compared to other members including this gene family (Fig 6B).

View larger version (25K): In this window In a new window Download PPT slide   Figure 5. mK6irs1/Krt2-6g gene mutations in five Ca alleles and the ENU-induced wavy coat mutant, M100689. (A) A schematic illustration of the genomic structure of the mouse mK6irs1/Krt2-6g gene depicts the translation initiation codon, exon numbers, and a stop codon. (B and C) Sequence analysis of exon 1 (B) and exon 7 (C) in wild type, Ca alleles, and M100689. Asterisks mark the mutation positions. (B) Heterozygous and homozygous 418-AAC-420 deletion found in CaRin, CaJ, Ca9J, and Ca10J. (C) Heterozygous 1271T G (L424W) missense substitution and homozygous 1292C to A (A431D) missense substitutions found in M100689 and CamedJ, respectively.

View larger version (63K): In this window In a new window Download PPT slide   Figure 6. mK6irs1/Krt2-6g is mutated in Ca mice. (A) Amino acid sequence of the mK6irs1/Krt2-6g gene. The central -helical rod domain is shown as an open box, the subdomains of which are marked by double-headed arrows. The amino acid deletion in CaRin, CaJ, Ca9J, and Ca10J is indicated by an arrow, and the point mutation in CamedJ and the ENU-induced wavy coat mutant, M100689, is marked with an asterisk. (B) Alignment of protein sequence in the human and mouse type II epithelial keratins. Arrow (deletion in CaRin, CaJ, Ca9J, and Ca10J) and asterisks (point mutation in CamedJ and M100689) mark the mutation positions. Both mutations occur in the highly conserved sequence of the -helical rod domain of this protein. The amino acid sequences of the -helical rod domain used for multiple alignments of the type II epithelial keratins of mouse (m) and human (h) were derived from the following cDNA sequence of the GenBank/EMBL database: mK6irs/Krt2-6g (AOKI et al. 2001 , accession no. NM_019956); hK6irs/Krt2-6g (LANGBEIN et al. 2001 , NM_033448); mK2-17 (HERZOG et al. 1994 , X74784); hK2e (COLLIN et al. 1992A , AF019084); mK2-1 (STEINERT et al. 1985 , NM_008473); hK1 (STEINERT et al. 1985 , NM_006121); mK2-4 (KNAPP et al. 1986 , NM_008475); hK4 (LEUBE et al. 1988 , NM_002272); hK2p (COLLIN et al. 1992B , Q01546); hK3 (MOLL et al. 1982 , NM_057088); mK2-6a (TAKAHASHI et al. 1998 , NM_008476); mK2-6b (TAKAHASHI et al. 1998 , NM_010669); hK6a (TAKAHASHI et al. 1995 , NM_005554); hK6b (TAKAHASHI et al. 1995 , NM_005555); mK6hf (POIRIER et al. 2002 , AF343088); hK6hf (WINTER et al. 1998 , NM_004693); mK2-5 (CARNINCI and HAYASHIZAKI 1999 , NM_027011); hK5 (LERSCH and FUCHS 1988 , NM_000424); mK2-7 (CARNINCI and HAYASHIZAKI 1999 , NM_033073); hK7 (GLASS and FUCHS 1988 , NM_005556); mK2-8 (VASSEUR et al. 1985 , NM_031170); and hK8 (LEUBE et al. 1986 , NM_002273). Multiple sequence alignment was performed using the CLUSTAL W program.

To identify mutations in the other Ca alleles, Ca^J, Ca^medJ, Ca^9J, Ca^10J, and Ca^Sl, we undertook genomic sequencing of the mK6irs1/Krt2-6g gene using DNA purchased from the Jackson Laboratory. Using a combination of long-range PCR with direct sequencing, we determined the nucleotide sequence of a 9.3-kb genomic DNA containing all exons and introns of the mK6irs1/Krt2-6g gene (GenBank accession nos. AB100414, AB100415, AB100416, AB100417, AB100418). Nucleotide sequence comparison revealed the three nucleotide deletions in exon 1 of Ca^J, Ca^9J, and Ca^10J to be completely identical to the deletion found in Ca^Rin (Fig 5). A different mK6irs1/Krt2-6g mutation was identified in Ca^medJ, a C to A conversion at exon 7 predicted to substitute aspartic acid for alanine at position 431 (Fig 5 and Fig 6A). At the same position of the murine Krt2-8 gene, an A to T amino acid substitution was discovered (Fig 6B). To date, we do not know whether the substitution is a mutation causing a phenotype change. Further studies, such as transgenesis of genomic clones derived from the Ca^medJ mutant, will clarify this issue. On the last allele, Ca^Sl, we could not detect any mutations in the mK6irs1/Krt2-6g gene with this technique.

Further, we have carried out the same analysis in two ENU-induced wavy hair mutants. One point mutation was identified in one mutant, M100689, a T to G transversion in exon 7 predicted to result in the substitution of tryptophan for leucine at amino acid position 424 (Fig 5 and Fig 6A). This leucine at 424 is also highly conserved among other epithelial keratin genes in mouse and human (Fig 6B). In another mutant, M100573, we could not detect any mutations in the mK6irs1/Krt2-6g gene with this approach.

To examine the effect of the Ca^Rin mutation on mK6irs1/Krt2-6g RNA expression, we carried out Northern blot and RT-PCR analysis using RNA isolated from the skin of 5-week-old +/+ and Ca^Rin/Ca^Rin mice. We selected this source because AOKI et al. 2001 had reported that of the major murine tissues, mK6irs1/Krt2-6g expression was found only in skin and was highest at the anagen stage. The level of mK6irs1/Krt2-6g expression was markedly reduced in Ca^Rin by both Northern blot and RT-PCR analysis (Fig 7).

View larger version (33K): In this window In a new window Download PPT slide   Figure 7. Northern blot (A) and RT-PCR (B) analyses of K6irs/Krt2-6g. Total RNA was obtained from 5-week-old skin. (A) K6irs/Krt2-6g expression levels were reduced in CaRin. The primary K6irs/Krt2-6g RNA is 2.2 kb. The blot was subsequently rehybridized with Gapdh. (B) To detect K6irs/Krt2-6g-specific transcripts, cDNA from B6 and B6-CaRin skin was screened with the primers K6irs7F and K6irs4R located in different exons. cDNA was amplified as a 750-bp K6irs/Krt2-6g product (top arrow; Table 2). The 1-kb Gapdh control band is also indicated (bottom).

AOKI et al. 2001 reported that mK6irs1/Krt2-6g is expressed in the Huxley and Henle layers of mouse inner hair sheath. To examine whether the expression pattern of mK6irs1/Krt2-6g is also retained in the Ca^Rin mutant mice, we compared the localization of the mK6IRS1/KRT2-6G protein in the hair follicle between the mutant and wild-type mice (Fig 8). As AOKI et al. 2001 reported, the mK6IRS1/KRT2-6G protein was expressed exclusively in the inner root sheath (Fig 8, A–C) in wild-type mice. On the other hand, the specificity of the mK6IRS1/KRT2-6G protein did evidently decrease in the Ca^Rin mutant mice (Fig 8, D–F). For example, the inner root sheath of the mutant mice was most strongly stained but the majority of the hair shaft was also weakly stained. This suggests that dysregulated mK6IRS1/KRT2-6G protein localization occurred in the mutant mice.

View larger version (85K): In this window In a new window Download PPT slide   Figure 8. Immunohistochemical comparison using anti-K6IRS/KRT2-6G polyclonal antibody between wild-type and mutant CaRin mice. Tissue distribution of K6IRS/KRT2-6G protein in the wild-type (A and C) and CaRin (D and F) mouse hair follicles at 5 weeks is shown. Using the K6irs/Krt2-6g polyclonal antibody, K6IRS/KRT2-6G protein is detected with a distinct distribution in the IRS of hair follicles in the wild-type mouse (A and C), whereas it has a rather fuzzy distribution in the mutant CaRin mice (D and F). Phase-contrast microscopy in the same area of wild-type (B) and mutant mice (E) is also shown. Ep, epidermis; De, dermis; irs and ors, inner and outer root sheath, respectively. Bar in A and D, 100 µm; in C and F, 20 µm.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

A type II keratin gene, mK6irs1/Krt2-6g, as the candidate for Ca mutation:
Using a positional cloning approach, we discovered that a strong candidate for the gene affected by the caracul (Ca) mutation is a type II keratin gene, mK6irs1/Krt2-6g, because five independent Ca alleles exhibit mutations in the mK6irs1/Krt2-6g gene (Fig 5). The Ca^Rin allele contains a small in-frame 3-bp deletion at nucleotides 420–422 in the first exon of the mK6irs1/Krt2-6g gene and this deletion putatively causes a one-amino-acid (asparagine) deletion. This asparagine deletion is located in a highly conserved region in the 310-amino-acid coiled-coil rod domain (-helical domain; Fig 6A). The -helical domain functions to form heteropolymers consisting of a specific type I and a type II cytokeratin through interactions of these domains (HATZFELD and WEBER 1990 ). VASSAR et al. 1991 showed that transgenic mice expressing a deleterious allele of human K14 displayed dominant-negative inhibition of endogenous cytokeratin expression mediated via the mutated -helical domain functions, i.e.; these mice exhibited abnormalities in epidermal architecture and often died prematurely. Therefore, the pathobiology and biochemistry of the transgenic mice and their cultured keratinocytes bore resemblance to a group of genetic disorders known as epidermolysis bullosa simplex. As another example of associated pathology, transgenic mice carrying a dominant-negative Krt2-6a gene suffered severe blistering and neonatal death (WOJCIK et al. 1999 ). Because the Ca^Rin phenotype is dominant and deleterious for hair texture and IRS morphology (Fig 1 and Fig 2), it is very likely that the asparagine deletion causes an alteration in the mK6IRS1 coiled-coil domain and a resultant dominant-negative phenotype. Furthermore, the expression of mK6IRS1 mRNA is markedly decreased in the skin of Ca^Rin mice, even in the anagen phase, when mK6irs1/Krt2-6g message peaked in nonaffected littermates (Fig 7). Assuming that the level of mK6irs1/Krt2-6g transcription is proportional to translation, this finding suggests that stoichiometric lack of the mK6IRS1 protein occurs in the cells and subsequently causes the decreased availability of heteropolymers. The same is true for the Ca^medJ allele, where a single C to A transversion generated an amino acid substitution of an aspartic acid for an alanine at position 431, which is highly conserved within the -rod helical domain; i.e., all type II keratin genes examined contain an alanine residue at this position (Fig 6). The ENU-induced Ca allele can also be explained in the same way. It is suggested that this amino acid replacement destroys the tertiary structure of the molecule and consequently prevents protein-protein interactions between the other cytokeratin counterparts.

A small deletion hotspot in mK6irs1/Krt2-6g gene:
We examined five Ca mutations and found that four of these, the preexistent alleles Ca^J, Ca^9J, Ca^10J, and our allele Ca^Rin, shared the same small deletion (Fig 5 and Fig 6). Because these alleles were independently derived (see JAX catalog; http://www.jax.org/jaxmice), this suggests that a mutation hotspot causing a small deletion is present in the first exon of the mK6irs1/Krt2-6g gene. If so, this is the first description that such a hotspot is present in the mouse. In humans, such hotspots have been reported in the SMAD4 gene in juvenile polyposis patients (HOWE et al. 2002 ) and in the interferon gamma receptor 1 (IFNGR1) gene associated with dominant susceptibility to mycobacterial infection (JOUANGUY et al. 1999 ). To date, little is known about precise molecular mechanisms responsible for hotspots. However, two main mechanisms causing such deletions have been proposed, namely slippage of DNA polymerase and slipped mispairing (JOUANGUY et al. 1999 ; HOWE et al. 2002 ). Both events are thought to be caused by the interactions between specific DNA sequences such as direct repeats, inverted repeats, palindromes, or a small deletion consensus motif and enzymes for DNA replication (GRUNDY et al. 1991 ; KRAWCZAK and COOPER 1991 , KRAWCZAK and COOPER 1993 ). In this murine small deletion hotspot, we found two direct repeats of CAA at nucleotides 417–419 and 420–422, either of which is deleted in the mutations, in the first exon of the mK6irs1/Krt2-6g gene. Two consensus motifs TG (A/G) (A/G) (G/T) (A/C) or (A/G) also exist in the first exon and intron 1 (Fig 9). Taking this evidence together with the lack of an intervening and polypurine sequence (JOUANGUY et al. 1999 ), it is likely that slippage of DNA polymerase occurs in the mK6irs1/Krt2-6g gene.

View larger version (10K): In this window In a new window Download PPT slide   Figure 9. A hotspot of small deletions in the mouse K6irs/Krt2-6g gene. The region surrounding position 420 of the coding region in exon 1 is represented. One proposed mechanism of the deletion would be slippage of DNA polymerase through these repeat sequences. Inverted repeats are another mechanism that causes loops on a single DNA strand and they were indeed present, flanking the deletion in this case. Sequence motifs in the vicinity of position 420 that may account for the small deletion hotspot are represented. The small deletion consensus sequences are underlined.

Role of mK6irs1/Krt2-6g in hair formation:
To date, the mK6IRS1/KRT2-6G molecule is the protein that is expressed exclusively in the IRS, in particular, in the Henle and Huxley layers of the hair follicle in IRS (AOKI et al. 2001 ; PORTER et al. 2001 ; LANGBEIN et al. 2002 ), although previous reports claimed the expression of other type I and II cytokeratins in IRS, such as K1, K10, K13, and K16 (STARK et al. 1990 ). Furthermore, its expression is stage specific in the hair cell cycle, i.e., can be detected during the hair follicle growth phase, but hardly at all at any other stage (AOKI et al. 2001 ). We confirmed the restricted expression of the mK6IRS1/KRT2-6G protein in IRS and furthermore we discovered that the expression pattern is disturbed in the Ca^Rin mutant mice (Fig 8). These lines of evidence suggest that the mK6IRS1/KRT2-6G protein plays important roles in the development of hair and the maintenance of the cells in the IRS (AOKI et al. 2001 ; PORTER et al. 2001 ; LANGBEIN et al. 2002 ). The findings documented here that the mK6irs1/Krt2-6g gene is a strong candidate for that causing the Ca mutation shed new light on its possible function(s), because phenotype analyses of the Ca mutant indicate that it causes abnormal hair texture as well as abnormal morphology in the IRS (Fig 1 and Fig 2). Because the IRS surrounds the hardening hair fiber and the central hair-forming unit proper, the Ca mutation is considered as causing primarily a defect in IRS function. In particular, the evidence that the IRS of Ca mice lacked uniformity of thickness and in some follicles the IRS cells showed abnormal keratinization (Fig 2) supports this contention. These lines of evidence suggest that the IRS most likely functions as a "corset" or a "barrel" molding the hair into its optimal shape during its progression toward the skin surface. Consequently, defects in the IRS compromise the correct shaping of the hair fiber, thus creating a wavy type.

It has been reported that the cytokeratin genes are involved in several spontaneous skin diseases in humans (VASSAR et al. 1991 ; CHAPALAIN et al. 2002 ). To date, no such spontaneous diseases associated with cytokeratin mutations have been reported in mice. On the other hand, mice with manipulated cytokeratin genes do show abnormalities; e.g., animals expressing a deleterious allele of the human K14 gene in the basal epidermis developed a cutaneous phenotype bearing some resemblance to the human disorder epidermolysis bullosa simplex (VASSAR et al. 1991 ), and mice carrying a mutated Krt2-6a transgene suffered severe blistering and neonatal death (WOJCIK et al. 1999 ). These lines of evidence suggest that we may be able to generate a useful model for human skin diseases through the manipulation of cytokeratin genes in mice and, in addition, facilitate the understanding of the function(s) of the gene in the IRS. We are now trying to generate such gene-manipulated mice using the mK6irs1/Krt2-6g gene.

	FOOTNOTES

Sequence data from this article have been deposited with the DDBJ/EMBL/GenBank Data Libraries under accession nos. AB100413, AB100414, AB100415, AB100416, AB100417, AB100418.
¹ These authors contributed equally to this work.

	ACKNOWLEDGMENTS

We thank Satomi Yamada and Yayoi Wakita of The Tokyo Metropolitan Institute of Medical Science and Kimio Kobayashi, Aya Shimizu, and Junko Nagano of Riken GSC for technical assistance. We are also grateful to Yutaka Shimomura and Masaaki Ito of Niigata University of School of Medicine for giving us the anti-mK6IRS1/KRT2-6G polyclonal antibody and to Kiyokazu Morioka of The Tokyo Metropolitan Institute of Medical Science for valuable comments on hair morphology and development. This research was supported by a Grant-in-Aid for Scientific Research (13680919) from the Japanese Society for the Promotion of Science and by a Health Sciences Research Grant (Research on Eye and Ear Science, Immunology, Allergy, and Organ Transplantation) from the Ministry of Health, Labor, and Welfare of Japan.

Manuscript received January 30, 2003; Accepted for publication June 4, 2003.

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