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Genomic Organization of the Human Hairless Gene (HR) and Identification of a Mutation Underlying Congenital Atrichia in an Arab Palestinian Family
Genomics 56, 141–148 (1999) Article ID geno.1998.5699, available online at http://www.idealibrary.com on
Genomic Organization of the Human Hairless Gene (HR) and Identification of a Mutation Underlying Congenital Atrichia in an Arab Palestinian Family Wasim Ahmad, Abraham Zlotogorski,* Andrei A. Panteleyev, HaMut Lam, Mahmud Ahmad,† Muhammad Faiyaz ul Haque,† ,‡ Husein M. Abdallah,§ Laryssa Dragan, ¶ and Angela M. Christiano\ ,1 Department of Dermatology and \Department of Genetics and Development, Columbia University, New York, New York 10032; *Department of Dermatology, Hadassah University Hospital, Jerusalem, Israel; †Department of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan; ‡Division of Medical Genetics, Cedars-Sinai Research Institute, Los Angeles, California 90048; §Department of Public Health, Ramallah, Israel; and ¶Section of Dermatology, University of Chicago School of Medicine, Chicago, Illinois 60637 Received August 20, 1998; accepted November 23, 1998
Congenital atrichia is a rare form of hereditary human hair loss, characterized by the complete shedding of hair shortly after birth, together with the formation of papular lesions on the skin. Recently, we cloned the human homolog of the mouse hairless gene and identified pathogenic mutations in several families with inherited congenital atrichia. Here, we present the genomic organization of the human hairless gene (HGMW-approved symbol HR), which spans over 14 kb on chromosome 8p12 and is organized into 19 exons. In addition, we report the identification of a 22-bp deletion mutation in exon 3 of the hairless gene in a large consanguineous Arab Palestinian family from a village near Jerusalem, Israel. These findings extend the body of evidence implicating mutations in the hairless gene as an underlying cause of congenital atrichia in humans. © 1999 Academic Press
INTRODUCTION
Hair follicle morphogenesis and subsequent cycling in adult mammals constitute a complex process that requires a series of reciprocal epithelial–mesenchymal signals for the correct execution of an intricate program of developmental events. The initial message is derived from the dermis and instructs the overlying epidermis to thicken, forming a placode and then a downgrowth into the dermis, known as the hair plug. This is followed by a second signal from the epidermis that instructs the dermis to form the dermal papilla. Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession No. AF039196. 1 To whom correspondence should be addressed at Department of Dermatology, Columbia University, College of Physicians and Surgeons, 630 West 168th Street VC15-1526, New York, NY, 10032. Telephone: (212) 305-9565. Fax: (212) 305-7391. E-mail: amc65@ columbia.edu.
The dermal papilla then stimulates the division of overlying epithelially derived matrix cells in the hair plug. These cells divide rapidly and differentiate into either inner root sheath cells or hair shaft cells, depending upon their position relative to the longitudinal axis of the hair follicle (Hardy, 1992). While these events have been described extensively in model systems, the genes governing these processes are largely unknown. Hair growth proceeds in a cyclical fashion throughout life, having three defined phases, the first of which is known as anagen, the stage during which the follicle is regenerated and a new hair grows. In humans, each hair is governed by its own temporal cycle of independent growth, in contrast to mice, where all hairs are cycling synchronously (Hardy, 1992). At a genetically predetermined time in each species, the follicles enter catagen, where hair elongation ceases and the follicle regresses due to the decrease in proliferation of the matrix cells. During catagen, the dermal papilla remains intact, but undergoes several remodeling events, including degradation of the elaborate extracellular matrix that is deposited during anagen. At the close of catagen, the hair is loosely anchored in a matrix of keratin, with the dermal papilla residing below. Finally, the follicle enters a quiescent phase known as telogen, during which the hair is usually shed. At the end of the resting phase, the dermal papilla migrates upward toward the epidermal stem cells located in the bulge region of the outer root sheath and recruits them downward again to form the hair matrix, thereby initiating a new cycle of hair growth (Costarelis et al., 1990; Rochat et al., 1994). Currently, surprisingly little is known about the molecular control of the signals that regulate progression through the hair cycle. There are several forms of hereditary human hair loss, which may represent a dysregulation of hair fol-
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licle cycling (Hardy, 1992), yet the molecular basis of these disorders has remained largely unexplored. Congenital atrichia (MIM 209500) is a rare form of complete hair loss together with papular skin lesions and is inherited in an autosomal recessive fashion (Landes and Langer, 1956; Cantu et al., 1980). In 1989, it was proposed that the hairless (hr) and rhino (hr rh) mouse mutations, which are allelic and map to mouse chromosome 14, bore striking resemblance to the human disease papular atrichia (Sundberg et al., 1989). We and others recently reported linkage of this phenotype to chromosome 8p12 (Ahmad et al., 1998a; Nothen et al., 1998), and we cloned and subsequently identified mutations in the human homolog of the mouse hairless gene in several large atrichia families from different regions of the world (Ahmad et al., 1998a,b; Zlotogorski et al., 1998), as well as in several alleles of hairless and rhino mice (Ahmad et al., 1998c,d; Panteleyev et al., 1998a). Cases resembling this disease from Pakistan, with loss of hair over the entire body, were recently reported under the name “congenital alopecia universalis” (OMIM 203655, Ahmad et al., 1993, 1998a; Nothen et al., 1998); however, “congenital atrichia with papules” (OMIM 209500) may represent a more precise description of the phenotype resulting from mutations in the human hairless gene. The hairless gene product is a putative transcription factor with a single zincfinger domain, which is highly expressed in brain and skin (Cachon-Gonzalez et al., 1994; Thompson, 1996; Ahmad et al., 1998a). It appears to function in the cellular transition to the first adult hair cycle, and in its absence, hair follicles disintegrate and a new hair is never induced (Panteleyev et al., 1998b,c,d). The result is the complete form of inherited hair loss observed in congenital atrichia. Here, we present the genomic organization of the human hairless gene, 2 which spans over 14 kb on chromosome 8p12 and is organized into 19 exons. In addition, we report the identification of a complex 22-bp deletion mutation in exon 3 of the hairless gene in a large Arab Palestinian family. These findings extend the body of evidence implicating mutations in the hairless gene as an underlying cause of congenital atrichia in humans. MATERIALS AND METHODS Identification of a genomic clone containing the human hairless gene. A BAC genomic clone (22-0-12) containing the human hairless gene was obtained using a commercially available human BAC library screening service (Research Genetics Inc.) with the primers 59-TGAGGGCTCTGTCCTCCTGC-39 (sense) and 59-GCTGGCTCCCTGGTGGTAGA-39 (antisense), designed to amplify exon 15 of the human HR gene (Ahmad et al., 1998a). BAC DNA was prepared using a ProPrep Plasmid Nucleic Acid Purification Kit with ProCipitate (LigoChem, Inc. Fairfield, NJ). Using a range of primer pairs designed from the published human hairless cDNA sequence (Ah-
2
The HGMW-approved symbol for the gene described in this paper is HR.
mad et al., 1998a; GenBank Accession No. AFO39196) and the genomic structure of the mouse gene (Cachon-Gonzalez et al., 1994), PCRs were performed to amplify segments of BAC DNA containing the human hairless exons. PCR was carried out for 35 cycles: 95°C for 1 min, annealing temperature for 1 min, and 72°C for 1 min, in an OmniGene Thermal Cycler (Marsh Scientific, Rochester, NY) in a reaction containing 100 ng of each primer, 13 buffer (Gibco BRL, Gaithersburg, MD), 50 ng of BAC DNA, 0.2 mM dNTPs, and 1.25 units Platinum Taq DNA polymerase (Gibco BRL) in each 50-ml reaction. The PCR products were electrophoresed in a 1–2% agarose gel in 13TBE buffer. The PCR products were eluted from the agarose gel with the QIAquick gel extraction kit (Qiagen Inc., Santa Clarita, CA) and sequenced on both strands using the ABI Prism dRhodamine Terminator Cycle Sequencing Ready Reaction Sequencing Kit and an ABI Model 310 DNA Sequencer (PE Applied Biosystems, Foster City, CA). When necessary, subcloning was performed into the BlueScript KSII vector (Stratagene, La Jolla, CA) according to the manufacturer’s recommendations. 39 Rapid amplification of cDNA ends (RACE) and transcript analysis. To determine the 39 end of the hairless cDNA, 39 RACE was performed using a Marathon Ready cDNA Amplification Kit (Clontech, Palo Alto, CA). The 59 forward primer (59-TCAGCGTCACTCAGCACTTCCTCTC-39) for the RACE PCR was derived from exon 18, and the reaction product was cloned into the TA cloning vector (Invitrogen, San Diego, CA). Plasmid DNA was prepared from five different clones using Qiagen columns (Qiagen Inc.) and sequenced as above. The Human Master Dot Blot was obtained from Clontech and hybridized using ExpressHyb solution according to the manufacturer’s recommendations, with a probe spanning exons 13–18 of the hairless cDNA, generated by RT-PCR from human skin fibroblast total mRNA, as previously described (Ahmad et al., 1998a). Nucleic acid preparation and genotyping. DNA was prepared from peripheral blood leukocytes according to standard techniques (Sambrook et al., 1989). For genotyping, one primer from each pair was labeled with [g- 33P]dATP (NEN, Boston MA). The PCR for each marker was performed in a 10 ml volume containing 50 ng of DNA, 50 ng of each primer, 200 mM dNTP, 13 PCR buffer (Gibco BRL) and one unit Platinum Taq DNA polymerase (Gibco BRL). PCR was carried out for 35 cycles: 95°C for 1 min, 58°C for 1 min, and 72°C for 1 min, in an OmniGene Thermal Cycler (Marsh Scientific). PCR products were electrophoresed on 6% denaturing polyacrylamide gels, and genotypes were assigned by visual inspection. Mutation detection. Exons and splice junctions were PCR amplified from genomic DNA and sequenced directly in an ABI Prism 310 Automated Sequencer, using the ABI Prism dRhodamine Terminator Cycle Sequencing Ready Reaction Sequencing Kit (PE Applied Biosystems), following purification in Centriflex Gel Filtration Cartridges (Edge Biosystems, Gaithersburg, MD). Exon 3 of the hairless gene was amplified with the following two sets of overlapping primers A and B, amplifying ;600- and 500-bp fragments, respectively: Primer set A, 59-GGCTTCAGTATTCTCCCCTT-39 (sense primer), and 59-TAGTGGGTGGGTAGGATGAA-39 (antisense primer); Primer set B, 59-CCTTGTTCATACTCTTGGCA-39 (sense primer), and 59GTGGTCCACTCATAAAGCCT-39 (antisense primer). Identification of the mutation was performed by visual comparison of the patients’ sequence with that of an unrelated, unaffected control individual.
RESULTS
Genomic Organization of Human Hairless Gene A single clone containing the hairless gene was isolated from a human genomic BAC library. The position and size of each exon and intron was determined by direct PCR amplification from the BAC clone, followed by automated sequencing (Fig. 1). The sequences of the intron– exon borders for the 19 exons of the human hairless gene are shown in Table 1. All sites conform to
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FIG. 1. Genomic organization of the human hairless gene. The gene contains 19 exons spanning 14 kb. The sizes of the exons are represented by proportional vertical bars, and the introns are represented by the horizontal line. Exon 1 contains the 59 UTR, exon 2 contains the initiating methionine, and exon 19 contains the 39 end of the coding sequence, the termination codon, and the 39 UTR. The zinc-finger domain in exon 6 is highlighted, as well as the LXXLL motifs in exons 5 and 10. Scale bars for intron and exon sizes are indicated on the lower right.
the mammalian consensus sequences for 59 donor and 39 acceptor splice sites. To obtain the sequences upstream of exon 2, the BAC DNA was digested and subcloned into the BlueScript KSII vector (Stratagene) and sequenced in the 59 direction upstream of exon 2. To identify putative cis-regulatory elements within the 59-flanking DNA, 1058 bp of genomic DNA upstream from exon 1 was sequenced. The results were analyzed using the program Signal, which compared the hairless sequence with known eukaryotic cis-elements. The sequences 59 to exon 1 contain several transcription factor-binding sites, including GATA-1, GATA-2, SP1, and AP1. Notably, the 59 flanking region has a high G1C content and lacks consensus TATA sequences. The initiating methionine residue is contained with exon 2. To determine the 39 end of the hairless mRNA, 39 RACE was performed using the AP1 primer and a 59 sense primer from exon 18 of the gene. An 850-bp PCR product was cloned and sequenced, which revealed 655-bp of noncoding sequence at the 39 end of the gene. A 10-kb subclone from the BAC clone, containing exons 18, 19, and the 39 end of the hairless gene, was sequenced to ensure that no additional intron was present in the 39 untranslated region. Exon 19 contains 60 bp of the open reading frame, the termination codon, and 655 bp of 39 untranslated region. All genomic sequences have been deposited with GenBank. Transcript Analysis A human RNA Master Blot filter containing immobilized human poly(A) 1 RNA from a wide range of tissues and developmental stages was hybridized with the hairless cDNA probe and showed the highest levels of gene expression in various parts of the brain. The gene was also found to be expressed at lower levels in colon, stomach, pituitary gland, salivary gland, small intestine, appendix, and fetal brain (Fig. 2). Clinical Findings To search for hairless gene mutations in atrichia patients, we studied six individuals from a large inbred
Arab Palestinian family originating from a village near Jerusalem, Israel. In this family, a total of 12 members, including 8 females and 4 males, were affected with congenital atrichia. The family pedigree is strongly suggestive of autosomal recessive inheritance with several consanguinity loops (Fig. 3A). Several members of the family, including an affected female individual (V-7), immigrated to the midwestern United States, and the pedigree was ascertained through this individual and traced back to Israel. The phenotypic appearance of congenital atrichia in this family is strikingly similar to that of patients reported in our previous studies (Ahmad et al., 1998a,b; Zlotogorski et al., 1998) and to that of patients described previously by others (Landes and Langer, 1956; Cantu et al., 1980; Ahmad et al., 1993). In affected individuals, hairs were typically absent from the scalp, and patients were almost completely devoid of eyebrows, eyelashes, axillary hair, and pubic hair (Individual V-7, Fig. 3B). In general, they are born with normal hair, which is shed during the first months to years of life and never regrows, similar to the pattern of hair loss observed in hairless mice (Sundberg, 1994). Affected individuals in this family also showed no growth or developmental delay, normal teeth and nails, and no abnormalities in sweating. Heterozygous members of the family had normal hair and were clinically indistinguishable from genotypically normal individuals in the family. A scalp skin biopsy from the affected individual showed a complete absence of hair follicles, and instead revealed deep dermal cysts and comedones reminiscent of those found in the skin of hairless mice (Montagna et al., 1952) (Fig. 3C). Collectively, the clinical and histopathological findings and pattern of inheritance were consistent with a diagnosis of congenital atrichia. Genotyping To determine whether affected individuals were homozygous for markers near the human hairless locus, genotyping of the six members of the family including two affected and four unaffected individuals was carried out for the polymorphic markers D8S1786 and
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TABLE 1 Intron/Exon Borders of the Human Hairless Gene Exon
Exon size
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
364 bp 652 bp 793 bp 151 bp 194 bp 165 bp 90 bp 116 bp 82 bp 164 bp 243 bp 166 bp 70 bp 131 bp 120 bp 116 bp 165 bp 129 bp 60 bp139UTR
Intron size 1223 727 .1 70 1524 770 183 799 388 99 208 168 212 500 97 1800 446 538
bp bp kb bp bp bp bp bp bp bp bp bp bp bp bp bp bp bp
39 Acceptor site — tatgctccagGGACCC ccctctttagGGCTTT gtcccctcagGACCCC ctctctccagATCGCC tggtcagcagGCCAAG tgccccacagGCTTTC ccacccccagCTTTGG ggtttaacagAAGGAA tccctgacagAGACCC ctctctgcagGATGAC cccaccacagCCTGTG cctcctgtagTTCGCC accctgacagGGTGGA tgccatgcagGTGTGA tcctcctcagACTTCC cttctttgagGTGTGC cccgacacagGTGCAG tttttcctagATGGAC
D8S298, closely linked to the hairless gene on chromosome 8p12 (Ahmad et al., 1998a). The markers were fully informative, and the two affected members of the family (IV-2 and V-7) were homozygous for both markers, suggesting linkage to the hairless locus. In addition, we found that all four unaffected individuals (IV29, V-4, V-5, and V-6) were carriers of the linked haplotype (data not shown). Taken together, the clini-
59 Donor site
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GCCCAGgtaagcgcca AGCAAGgtgagtgcag AGAAAGgtaagggggc GCGGAGgtgagccatt GGGAAGgtgaacagga AAGCAGgtaggagagg GCCAGGgtgagccatc CAGCAGgtaagaccca AAGAGGgtgagcggct CCCAGTgtgagtgag GGCCAGgtgaggcacc CTGAGCgtaagtgtcc CAGCAGgtgtgtatgt CCTATGgtgagtgtcc AGAAAGgtaggtcctc CAGATGgtgaggaggc CACCAGgtgctttcca GCCCAGgtgagtggga
cal presentation and suggestive linkage to chromosome 8p12 indicated that hairless was a strong candidate gene to analyze for an underlying mutation. Mutation Identification To screen for a mutation in the human hairless gene, exons and splice junctions were PCR amplified from genomic DNA and sequenced directly. Sequence analysis of exon 3 of the hairless gene from two affected individuals (IV-2 and V-7) revealed a homozygous complex 22-bp out-of-frame deletion consisting of a 1-bp (delC) at nucleotide position 1256 and a 21-bp (del21) extending from nucleotide position 1261–1281. Together, the deletions led to a frameshift and premature termination codon 77 bp downstream within exon 3 (Fig. 4). The mutation, designated 1256delC;1261del21, was present in the heterozygous state in the obligate carriers in the family. DISCUSSION
FIG. 2. Transcript analysis of the human hairless gene. Expression of hairless mRNA was noted in all areas of the brain including A1, whole brain; A2, amygdala; A3, caudate nucleus; A4, cerebellum; A5, cerebral cortex; A6, frontal lobe; A7, hippocampus; A8, medulla oblongata; B1, occipital lobe; B2, putamen; B3, substantia nigra; B4, temporal lobe; B5, thalamus; B6, subthalamic nucleus; B7, spinal cord; and G1, fetal brain. Lower levels of expression were noted in C4, colon; C8, stomach; D7, salivary gland; E3, small intestine; F1, appendix; and F3, trachea.
The genomic organization of the hairless gene is highly conserved between human and mouse. In addition, the amino acid sequences of murine and primate hairless are 85 and 95% identical to the human homolog, respectively (Ahmad et al., 1998a; and unpublished results). Comparison of the intron– exon positions of the human hairless gene with those of the mouse homolog shows almost exact conservation of the positioning of the introns. The gene spans over 14 kb on human chromosome 8p12 and is encoded by 19 exons ranging from 70 to 793 bp in size (Fig. 1). Similarly, no significant differences in intron size were observed between the human and the mouse hairless genes. Hairless is a putative single zinc-finger transcription factor, with only two significant regions of
FIG. 3. Clinical and histological findings in the family with papular atrichia. (A) Pedigree of the family with obligate carriers indicated with a dotted figure, and affected individuals as solid figures. Double lines represent consanguineous unions, and the proband (individual V-7) is indicated by an arrow. (B) Clinical appearance of papular atrichia in individual V-7. The patient is nearly devoid of scalp hairs and has sparse eyebrows and eyelashes. (C) Histological findings reveal the complete absence of normal hair follicle structures, which are instead replaced by residual epithelial cell conglomerates and large dermal cysts. No excessive perifollicular infiltrate is visible, and the sebaceous glands, sweat glands, and interfollicular epidermis are normal (hematoxylin & eosin, 1253 magnification).
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FIG. 4. Sequence analysis of the hairless gene. (Top) The wildtype sequence of exon 3. (Middle) Sequence analysis of a heterozygous carrier. (Bottom) The 22-bp deletion in the homozygous state in an affected individual. The arrow and bar above the wildtype sequence in the top panel represent the sequence that is deleted in the homozygous state in the patient in the bottom panel.
homology to any known proteins. First, the six-cysteine zinc-finger motif contained within exon 6 of the human and mouse hairless genes (amino acids 600 – 625) shares weak homology with TSGA, a testis-specific putative transcription factor of unknown function (Hoog et al., 1991). Supporting evidence for the functional importance of the zinc-finger domain comes from mutational studies in an Irish family with atrichia, in which affected individuals are homozygous for a missense mutation between the fourth and the fifth cysteines of the zinc-finger (Ahmad et al., 1998b). The second region of weak homology is found toward the 39 end of the gene, in the region extending from amino acids 704 to 1031 of the human sequence. In this region, significant homology is found between hairless and a family of proteins known as TRIPs (thyroid hormone receptor interacting proteins) (Lee et al., 1995). The signature motif (LXXLL) for TRIPs and other transcriptional coactivators is necessary and sufficient to mediate binding of this class of proteins to liganded
nuclear receptors (Heery et al., 1997). It is noteworthy that hairless contains two LXXLL motifs, one in exon 5 (LCRLL, amino acids 566 –570) and a second within the region of TRIP homology (LCELL, amino acids 758 –762), adding evidence in support of functional significance of this region of the gene. Further implication of the region spanning from 704 to 1031 in functional integrity of hairless comes from mutation analysis in a Pakistani atrichia family with a missense mutation in the same region (Ahmad et al., 1998a). This rare form of inherited human hair loss was named atrichia with papular lesions in 1950 and was characterized by normal hair formation at birth followed by hair loss associated with the formation of comedones and follicular cysts (Fredrich, 1950). Later, in 1989, the human disease was first proposed as a homolog of the hairless mouse mutation (Sundberg et al., 1989). The molecular basis of the hairless mouse phenotype was shown to be the result of a murine leukemia proviral insertion into intron 6 of the hairless
HUMAN HAIRLESS GENE (HR) AND MUTATION IN CONGENITAL ATRICHIA
gene, resulting in aberrant splicing and a moderately severe phenotype (Cachon-Gonzalez et al., 1994). A second, phenotypically more severe allelic mutation, known as rhino, is the result of more deleterious nonsense and deletion mutations in the hairless gene (Ahmad et al., 1998c,d; Panteleyev et al., 1998a). Recently, the predicted connection between hairless mice and papular atrichia in humans was confirmed when mutations underlying this disorder were identified in the human hairless gene (Ahmad et al., 1998a,b; Zlotogorski et al., 1998). The mutation described in this study is the second reported deletion mutation in the human hairless gene. Since the mutation results in a frameshift and a downstream premature termination codon, we predict an absence of functional mRNA due to nonsense-mediated mRNA decay (Maquat, 1996) and consequently, an absence of hairless protein. Interestingly, however, in contrast to rhino and hairless mice, there appears to be no difference in phenotypic severity between human patients with different types of mutations in the hairless gene. It is intriguing that despite the high levels of hairless gene expression in the brain, neither rhino or hairless mice, nor any of the human atrichia patients we have studied, exhibit any detectable neurological phenotype or developmental delay reflecting aberrant hairless expression in developing CNS. The findings presented in this study extend the body of evidence implicating different types of mutations in the hairless gene as the underlying cause of congenital atrichia in humans. Previously, it was shown that hairless gene expression is primarily restricted to the skin and brain (Cachon-Gonzalez et al., 1994; Thompson, 1996; Ahmad et al., 1998a). To more fully characterize the sites of hairless expression, a dot blot containing poly(A) 1 RNA from several human tissues at different developmental stages was hybridized with the human hairless cDNA probe. In addition to high levels of gene expression detected in various parts of the brain, lower levels of hairless gene expression were also observed in colon, stomach, pituitary gland, salivary gland, small intestine, and appendix (Fig. 2). Although the precise function of hairless in the brain is still elusive, hairless protein has been shown to interact directly and specifically with thyroid hormone receptor, the same protein that induces its expression. Thus, in the brain, hairless appears to function both as a downstream target and as an upstream regulator of thyroid hormone action, potentially as a transcriptional corepressor (Thompson, 1996; Thompson and Bottcher, 1997). Further, a thyroid hormone response element (TRE) was identified 9 kb upstream of the transcriptional start site in the rat hairless gene. The TRE in the human and mouse hairless genes has not been identified as yet, and it remains to be determined whether hairless is similarly regulated by thyroid hormone in the skin. Interestingly, when hairless cDNA was amplified from human skin fibroblast total mRNA for generating the probe for the dot blot, we noted the
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consistent and complete absence of exon 17 from several independent RT-PCR products. This finding suggests that hairless may be subject to tissue-specific alternative splicing events, which may in turn specify differential cell-type specific functions. The cellular events leading to the development of hairlessness involve a premature and massive apoptosis in the hair matrix cells, together with a concomitant decline in Bcl-2 expression, a loss of NCAM positivity, and a disconnection with the overlying epithelial sheath essential for the movement of the dermal papilla (Panteleyev et al., 1998b,c,d). As a consequence, the dermal papilla remains stranded in the dermis, and indispensable messages between the dermal papilla and stem cells in the bulge are not transmitted, thus no further hair growth occurs. In hairless mice and in humans with congenital atrichia, we postulate that the absence of functional hairless protein leads to initiation of a premature and aberrant catagen phase due to abnormal apoptosis, dysregulation of cell adhesion, and defects in dermal papilla-derived signaling that normally control catagen-associated hair follicle remodeling (Panteleyev et al., 1998b,c,d). These observations suggest that a crucial role of the hairless protein may be involved in maintaining the balance between cell proliferation, differentiation, and apoptosis during hair follicle cycling. The identification of mutations in the hairless gene in humans and mice with congenital atrichia underscores a crucial role of the hairless gene product in the regulation of hair growth. Delineation of the genomic organization of the human hairless gene will not only facilitate the identification of mutations in patients with congenital atrichia, but also advance functional studies into the role of the hairless gene product in hair follicle morphogenesis, hair growth, and hair cycling. ACKNOWLEDGMENTS We appreciate the generous participation of the family members in this study. This work was supported in part by grants from the National Alopecia Areata Foundation (A.Z. and A.M.C.) and the NIH-NIAMS Skin Disease Research Center (P30-AR44535) in the Department of Dermatology at Columbia University.
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Ahmad, W., Panteleyev, A., Sundberg, J. P., and Christiano, A. M. (1998c). Molecular basis for the rhino (hr rh8J ) phenotype: A nonsense mutation in the mouse hairless gene. Genomics 53: 383–386 Ahmad, W., Panteleyev, A. A., Henson-Apollonio, V., Sundberg, J. P., and Christiano, A. M. (1998d). Molecular basis of a novel rhino (hr rhChr) phenotype: A nonsense mutation in the mouse hairless gene. Exp. Dermatol. 7: 298 –301. Cachon-Gonzalez, M. B., Fenner, S., Coffin, J. M., Moran, C., Best, S., and Stoye, J. P. (1994). Structure and expression of the hairless gene of mice. Proc. Natl. Acad. Sci. USA 91: 7717– 7721. Cantu, J. M., Sanchez-Corona, J., Gonzalez-Mendoza, A., Martinez, Y., Martinez, R., and Garcia-Cruz, D. (1980). Autosomal recessive inheritance of atrichia congenita. Clin. Genet. 17: 209 –212. Costarelis, G., Sun, T.-T., and Lavker, R. M. (1990). Label retaining cells reside in the bulge area of pilosebaceous unit: Implications for follicular stem cells, hair cycle, and skin carcinogenesis. Cell 61: 1329 –1337. Fredrich, H. C. (1950). Zur kenntnis der kongenitale hypotrichosis. Dermatol. Wochenschr. 121: 408 – 410. Hardy, M. (1992). The secret life of the hair follicle. Trends Genet. 8: 55– 61. Heery, D. M., Kalkhoven, E., Hoare, S., and Parker, M. G. (1997). A signature motif in transcriptional co-activators mediates binding to nuclear receptors. Nature 387: 733–736. Hoog, C., Schalling, M., Grunder-Brundell, E., and Daneholt, B. (1991). Analysis of a murine male germ cell-specific transcript that encodes a putative zinc finger protein. Mol. Reprod. Dev. 30: 173– 181. Landes, E., and Langer, I. (1956). Ein beitrag zur hypotrichosis congenita. Hautzart 7: 413– 415. Lee, J. W., Choi, H.-S., Gyuris, J., Brent, R., and Moore, D. D. (1995). Two classes of proteins dependent on either the presence or absence of thyroid hormone for interaction with the thyroid hormone receptor. Mol. Endocrinol. 9: 243–254. Maquat, L. E. (1996). Defects in RNA splicing and the consequence of shortened translational reading frames. Am. J. Hum. Genet. 59: 279 –286. Montagna, W., Chase, H. B., and Melaragno, H. P. (1952). The skin of hairless mice. The formation of cysts and the distribution of lipids. J. Invest. Dermatol. 19: 83–94. Nothen, M. M., Cichon, S., Vogt, I. R., Hemmer, S., Kruse, R., Knapp, M., Holler, T., Ul Haque, M. F., Ul Haque, S., Propping, P., Ahmad, M., and Rietschel, M. (1998). A gene for autosomal congenital
alopecia maps to chromosome 8p21–22. Am. J. Hum. Genet. 62: 386 –390. Panteleyev, A. A., Ahmad, W., Malashenko, A. M., Ignatieva, E. L., Paus, R., Sundberg, J. P., and Christiano, A. M. (1998a). Molecular basis for the rhino Yurlovo (hr rhY ) phenotype: Severe skin abnormalities and female reproductive defects associated with an insertion in the hairless gene. Exp. Dermatol. 7: 281–288. Panteleyev, A. A., van der Veen, C., Rosenbach, T., Muller-Rover, S., Sokolov, V. E., and Paus, R. (1998b). Towards defining the pathogenesis of the hairless phenotype. J. Invest. Dermatol. 110: 903– 907. Panteleyev, A. A., Botchkareva, N. V., van der Veen, C., Christiano, A. M., and Paus, R. (1998c). Pathobiology of the hairless phenotype: Dysregulation of hair follicle apoptosis and topobiology during the initiation of follicle cycling. J. Invest. Dermatol. 110: 577. Panteleyev, A. A., Paus, R., Ahmad, W., Sundberg, J. P., and Christiano, A. M. (1998d). Molecular and functional aspects of the hairless (hr) gene in laboratory rodents and humans. Exp. Dermatol. 7: 249 –267. Rochat, A., Kobayashi, K., and Barrandon, Y. (1994). Location of stem cells in human hair follicles by clonal analysis. Cell 76: 1063–1073. Sambrook, J., Fritsch, E. G., and Maniatis, T. (1989). “Molecular Cloning: A Laboratory Manual,” 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Sundberg, J. P. (1994). The hairless (hr) and rhino (hr rh ) mutations, chromosome 14. In “Handbook of Mouse Mutations with Skin and Hair Abnormalities: Animal Models and Biomedical Tools” (J. P. Sundberg, Ed.), pp. 291–312, CRC Press, Boca Raton, FL. Sundberg, J. P., Dunstan, R. W., and Compton, J. G. (1989). Hairless mouse, HRS/J hr/hr. In “Monographs on Pathology of Laboratory Animals. Integument and Mammary Glands” (T. C. Jones, U. Mohr, and R. D. Hunt, Eds.), pp. 192–197, Springer-Verlag, Heidelberg. Thompson, C. C. (1996). Thyroid hormone-responsive genes in developing cerebellum include a novel synaptotagmin and a hairless homolog. J. Neurosci. 16: 7832–7840. Thompson, C. C., and Bottcher, M. C. (1997). The product of a thyroid hormone-responsive gene interacts with thyroid hormone receptors. Proc. Natl. Acad. Sci. USA 94: 8527– 8532. Zlotogorski, A., Ahmad, W., and Christiano, A. M. (1998). Congenital atrichia in five Arab Palestinian families resulting in a mutation in the human hairless gene. Hum. Genet. 103: 400 – 404.
Genomic Organization of the Human Hairless Gene (HR) and Identification of a Mutation Underlying Congenital Atrichia in an Arab Palestinian Family Wasim Ahmad, Abraham Zlotogorski,* Andrei A. Panteleyev, HaMut Lam, Mahmud Ahmad,† Muhammad Faiyaz ul Haque,† ,‡ Husein M. Abdallah,§ Laryssa Dragan, ¶ and Angela M. Christiano\ ,1 Department of Dermatology and \Department of Genetics and Development, Columbia University, New York, New York 10032; *Department of Dermatology, Hadassah University Hospital, Jerusalem, Israel; †Department of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan; ‡Division of Medical Genetics, Cedars-Sinai Research Institute, Los Angeles, California 90048; §Department of Public Health, Ramallah, Israel; and ¶Section of Dermatology, University of Chicago School of Medicine, Chicago, Illinois 60637 Received August 20, 1998; accepted November 23, 1998
Congenital atrichia is a rare form of hereditary human hair loss, characterized by the complete shedding of hair shortly after birth, together with the formation of papular lesions on the skin. Recently, we cloned the human homolog of the mouse hairless gene and identified pathogenic mutations in several families with inherited congenital atrichia. Here, we present the genomic organization of the human hairless gene (HGMW-approved symbol HR), which spans over 14 kb on chromosome 8p12 and is organized into 19 exons. In addition, we report the identification of a 22-bp deletion mutation in exon 3 of the hairless gene in a large consanguineous Arab Palestinian family from a village near Jerusalem, Israel. These findings extend the body of evidence implicating mutations in the hairless gene as an underlying cause of congenital atrichia in humans. © 1999 Academic Press
INTRODUCTION
Hair follicle morphogenesis and subsequent cycling in adult mammals constitute a complex process that requires a series of reciprocal epithelial–mesenchymal signals for the correct execution of an intricate program of developmental events. The initial message is derived from the dermis and instructs the overlying epidermis to thicken, forming a placode and then a downgrowth into the dermis, known as the hair plug. This is followed by a second signal from the epidermis that instructs the dermis to form the dermal papilla. Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession No. AF039196. 1 To whom correspondence should be addressed at Department of Dermatology, Columbia University, College of Physicians and Surgeons, 630 West 168th Street VC15-1526, New York, NY, 10032. Telephone: (212) 305-9565. Fax: (212) 305-7391. E-mail: amc65@ columbia.edu.
The dermal papilla then stimulates the division of overlying epithelially derived matrix cells in the hair plug. These cells divide rapidly and differentiate into either inner root sheath cells or hair shaft cells, depending upon their position relative to the longitudinal axis of the hair follicle (Hardy, 1992). While these events have been described extensively in model systems, the genes governing these processes are largely unknown. Hair growth proceeds in a cyclical fashion throughout life, having three defined phases, the first of which is known as anagen, the stage during which the follicle is regenerated and a new hair grows. In humans, each hair is governed by its own temporal cycle of independent growth, in contrast to mice, where all hairs are cycling synchronously (Hardy, 1992). At a genetically predetermined time in each species, the follicles enter catagen, where hair elongation ceases and the follicle regresses due to the decrease in proliferation of the matrix cells. During catagen, the dermal papilla remains intact, but undergoes several remodeling events, including degradation of the elaborate extracellular matrix that is deposited during anagen. At the close of catagen, the hair is loosely anchored in a matrix of keratin, with the dermal papilla residing below. Finally, the follicle enters a quiescent phase known as telogen, during which the hair is usually shed. At the end of the resting phase, the dermal papilla migrates upward toward the epidermal stem cells located in the bulge region of the outer root sheath and recruits them downward again to form the hair matrix, thereby initiating a new cycle of hair growth (Costarelis et al., 1990; Rochat et al., 1994). Currently, surprisingly little is known about the molecular control of the signals that regulate progression through the hair cycle. There are several forms of hereditary human hair loss, which may represent a dysregulation of hair fol-
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licle cycling (Hardy, 1992), yet the molecular basis of these disorders has remained largely unexplored. Congenital atrichia (MIM 209500) is a rare form of complete hair loss together with papular skin lesions and is inherited in an autosomal recessive fashion (Landes and Langer, 1956; Cantu et al., 1980). In 1989, it was proposed that the hairless (hr) and rhino (hr rh) mouse mutations, which are allelic and map to mouse chromosome 14, bore striking resemblance to the human disease papular atrichia (Sundberg et al., 1989). We and others recently reported linkage of this phenotype to chromosome 8p12 (Ahmad et al., 1998a; Nothen et al., 1998), and we cloned and subsequently identified mutations in the human homolog of the mouse hairless gene in several large atrichia families from different regions of the world (Ahmad et al., 1998a,b; Zlotogorski et al., 1998), as well as in several alleles of hairless and rhino mice (Ahmad et al., 1998c,d; Panteleyev et al., 1998a). Cases resembling this disease from Pakistan, with loss of hair over the entire body, were recently reported under the name “congenital alopecia universalis” (OMIM 203655, Ahmad et al., 1993, 1998a; Nothen et al., 1998); however, “congenital atrichia with papules” (OMIM 209500) may represent a more precise description of the phenotype resulting from mutations in the human hairless gene. The hairless gene product is a putative transcription factor with a single zincfinger domain, which is highly expressed in brain and skin (Cachon-Gonzalez et al., 1994; Thompson, 1996; Ahmad et al., 1998a). It appears to function in the cellular transition to the first adult hair cycle, and in its absence, hair follicles disintegrate and a new hair is never induced (Panteleyev et al., 1998b,c,d). The result is the complete form of inherited hair loss observed in congenital atrichia. Here, we present the genomic organization of the human hairless gene, 2 which spans over 14 kb on chromosome 8p12 and is organized into 19 exons. In addition, we report the identification of a complex 22-bp deletion mutation in exon 3 of the hairless gene in a large Arab Palestinian family. These findings extend the body of evidence implicating mutations in the hairless gene as an underlying cause of congenital atrichia in humans. MATERIALS AND METHODS Identification of a genomic clone containing the human hairless gene. A BAC genomic clone (22-0-12) containing the human hairless gene was obtained using a commercially available human BAC library screening service (Research Genetics Inc.) with the primers 59-TGAGGGCTCTGTCCTCCTGC-39 (sense) and 59-GCTGGCTCCCTGGTGGTAGA-39 (antisense), designed to amplify exon 15 of the human HR gene (Ahmad et al., 1998a). BAC DNA was prepared using a ProPrep Plasmid Nucleic Acid Purification Kit with ProCipitate (LigoChem, Inc. Fairfield, NJ). Using a range of primer pairs designed from the published human hairless cDNA sequence (Ah-
2
The HGMW-approved symbol for the gene described in this paper is HR.
mad et al., 1998a; GenBank Accession No. AFO39196) and the genomic structure of the mouse gene (Cachon-Gonzalez et al., 1994), PCRs were performed to amplify segments of BAC DNA containing the human hairless exons. PCR was carried out for 35 cycles: 95°C for 1 min, annealing temperature for 1 min, and 72°C for 1 min, in an OmniGene Thermal Cycler (Marsh Scientific, Rochester, NY) in a reaction containing 100 ng of each primer, 13 buffer (Gibco BRL, Gaithersburg, MD), 50 ng of BAC DNA, 0.2 mM dNTPs, and 1.25 units Platinum Taq DNA polymerase (Gibco BRL) in each 50-ml reaction. The PCR products were electrophoresed in a 1–2% agarose gel in 13TBE buffer. The PCR products were eluted from the agarose gel with the QIAquick gel extraction kit (Qiagen Inc., Santa Clarita, CA) and sequenced on both strands using the ABI Prism dRhodamine Terminator Cycle Sequencing Ready Reaction Sequencing Kit and an ABI Model 310 DNA Sequencer (PE Applied Biosystems, Foster City, CA). When necessary, subcloning was performed into the BlueScript KSII vector (Stratagene, La Jolla, CA) according to the manufacturer’s recommendations. 39 Rapid amplification of cDNA ends (RACE) and transcript analysis. To determine the 39 end of the hairless cDNA, 39 RACE was performed using a Marathon Ready cDNA Amplification Kit (Clontech, Palo Alto, CA). The 59 forward primer (59-TCAGCGTCACTCAGCACTTCCTCTC-39) for the RACE PCR was derived from exon 18, and the reaction product was cloned into the TA cloning vector (Invitrogen, San Diego, CA). Plasmid DNA was prepared from five different clones using Qiagen columns (Qiagen Inc.) and sequenced as above. The Human Master Dot Blot was obtained from Clontech and hybridized using ExpressHyb solution according to the manufacturer’s recommendations, with a probe spanning exons 13–18 of the hairless cDNA, generated by RT-PCR from human skin fibroblast total mRNA, as previously described (Ahmad et al., 1998a). Nucleic acid preparation and genotyping. DNA was prepared from peripheral blood leukocytes according to standard techniques (Sambrook et al., 1989). For genotyping, one primer from each pair was labeled with [g- 33P]dATP (NEN, Boston MA). The PCR for each marker was performed in a 10 ml volume containing 50 ng of DNA, 50 ng of each primer, 200 mM dNTP, 13 PCR buffer (Gibco BRL) and one unit Platinum Taq DNA polymerase (Gibco BRL). PCR was carried out for 35 cycles: 95°C for 1 min, 58°C for 1 min, and 72°C for 1 min, in an OmniGene Thermal Cycler (Marsh Scientific). PCR products were electrophoresed on 6% denaturing polyacrylamide gels, and genotypes were assigned by visual inspection. Mutation detection. Exons and splice junctions were PCR amplified from genomic DNA and sequenced directly in an ABI Prism 310 Automated Sequencer, using the ABI Prism dRhodamine Terminator Cycle Sequencing Ready Reaction Sequencing Kit (PE Applied Biosystems), following purification in Centriflex Gel Filtration Cartridges (Edge Biosystems, Gaithersburg, MD). Exon 3 of the hairless gene was amplified with the following two sets of overlapping primers A and B, amplifying ;600- and 500-bp fragments, respectively: Primer set A, 59-GGCTTCAGTATTCTCCCCTT-39 (sense primer), and 59-TAGTGGGTGGGTAGGATGAA-39 (antisense primer); Primer set B, 59-CCTTGTTCATACTCTTGGCA-39 (sense primer), and 59GTGGTCCACTCATAAAGCCT-39 (antisense primer). Identification of the mutation was performed by visual comparison of the patients’ sequence with that of an unrelated, unaffected control individual.
RESULTS
Genomic Organization of Human Hairless Gene A single clone containing the hairless gene was isolated from a human genomic BAC library. The position and size of each exon and intron was determined by direct PCR amplification from the BAC clone, followed by automated sequencing (Fig. 1). The sequences of the intron– exon borders for the 19 exons of the human hairless gene are shown in Table 1. All sites conform to
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FIG. 1. Genomic organization of the human hairless gene. The gene contains 19 exons spanning 14 kb. The sizes of the exons are represented by proportional vertical bars, and the introns are represented by the horizontal line. Exon 1 contains the 59 UTR, exon 2 contains the initiating methionine, and exon 19 contains the 39 end of the coding sequence, the termination codon, and the 39 UTR. The zinc-finger domain in exon 6 is highlighted, as well as the LXXLL motifs in exons 5 and 10. Scale bars for intron and exon sizes are indicated on the lower right.
the mammalian consensus sequences for 59 donor and 39 acceptor splice sites. To obtain the sequences upstream of exon 2, the BAC DNA was digested and subcloned into the BlueScript KSII vector (Stratagene) and sequenced in the 59 direction upstream of exon 2. To identify putative cis-regulatory elements within the 59-flanking DNA, 1058 bp of genomic DNA upstream from exon 1 was sequenced. The results were analyzed using the program Signal, which compared the hairless sequence with known eukaryotic cis-elements. The sequences 59 to exon 1 contain several transcription factor-binding sites, including GATA-1, GATA-2, SP1, and AP1. Notably, the 59 flanking region has a high G1C content and lacks consensus TATA sequences. The initiating methionine residue is contained with exon 2. To determine the 39 end of the hairless mRNA, 39 RACE was performed using the AP1 primer and a 59 sense primer from exon 18 of the gene. An 850-bp PCR product was cloned and sequenced, which revealed 655-bp of noncoding sequence at the 39 end of the gene. A 10-kb subclone from the BAC clone, containing exons 18, 19, and the 39 end of the hairless gene, was sequenced to ensure that no additional intron was present in the 39 untranslated region. Exon 19 contains 60 bp of the open reading frame, the termination codon, and 655 bp of 39 untranslated region. All genomic sequences have been deposited with GenBank. Transcript Analysis A human RNA Master Blot filter containing immobilized human poly(A) 1 RNA from a wide range of tissues and developmental stages was hybridized with the hairless cDNA probe and showed the highest levels of gene expression in various parts of the brain. The gene was also found to be expressed at lower levels in colon, stomach, pituitary gland, salivary gland, small intestine, appendix, and fetal brain (Fig. 2). Clinical Findings To search for hairless gene mutations in atrichia patients, we studied six individuals from a large inbred
Arab Palestinian family originating from a village near Jerusalem, Israel. In this family, a total of 12 members, including 8 females and 4 males, were affected with congenital atrichia. The family pedigree is strongly suggestive of autosomal recessive inheritance with several consanguinity loops (Fig. 3A). Several members of the family, including an affected female individual (V-7), immigrated to the midwestern United States, and the pedigree was ascertained through this individual and traced back to Israel. The phenotypic appearance of congenital atrichia in this family is strikingly similar to that of patients reported in our previous studies (Ahmad et al., 1998a,b; Zlotogorski et al., 1998) and to that of patients described previously by others (Landes and Langer, 1956; Cantu et al., 1980; Ahmad et al., 1993). In affected individuals, hairs were typically absent from the scalp, and patients were almost completely devoid of eyebrows, eyelashes, axillary hair, and pubic hair (Individual V-7, Fig. 3B). In general, they are born with normal hair, which is shed during the first months to years of life and never regrows, similar to the pattern of hair loss observed in hairless mice (Sundberg, 1994). Affected individuals in this family also showed no growth or developmental delay, normal teeth and nails, and no abnormalities in sweating. Heterozygous members of the family had normal hair and were clinically indistinguishable from genotypically normal individuals in the family. A scalp skin biopsy from the affected individual showed a complete absence of hair follicles, and instead revealed deep dermal cysts and comedones reminiscent of those found in the skin of hairless mice (Montagna et al., 1952) (Fig. 3C). Collectively, the clinical and histopathological findings and pattern of inheritance were consistent with a diagnosis of congenital atrichia. Genotyping To determine whether affected individuals were homozygous for markers near the human hairless locus, genotyping of the six members of the family including two affected and four unaffected individuals was carried out for the polymorphic markers D8S1786 and
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TABLE 1 Intron/Exon Borders of the Human Hairless Gene Exon
Exon size
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
364 bp 652 bp 793 bp 151 bp 194 bp 165 bp 90 bp 116 bp 82 bp 164 bp 243 bp 166 bp 70 bp 131 bp 120 bp 116 bp 165 bp 129 bp 60 bp139UTR
Intron size 1223 727 .1 70 1524 770 183 799 388 99 208 168 212 500 97 1800 446 538
bp bp kb bp bp bp bp bp bp bp bp bp bp bp bp bp bp bp
39 Acceptor site — tatgctccagGGACCC ccctctttagGGCTTT gtcccctcagGACCCC ctctctccagATCGCC tggtcagcagGCCAAG tgccccacagGCTTTC ccacccccagCTTTGG ggtttaacagAAGGAA tccctgacagAGACCC ctctctgcagGATGAC cccaccacagCCTGTG cctcctgtagTTCGCC accctgacagGGTGGA tgccatgcagGTGTGA tcctcctcagACTTCC cttctttgagGTGTGC cccgacacagGTGCAG tttttcctagATGGAC
D8S298, closely linked to the hairless gene on chromosome 8p12 (Ahmad et al., 1998a). The markers were fully informative, and the two affected members of the family (IV-2 and V-7) were homozygous for both markers, suggesting linkage to the hairless locus. In addition, we found that all four unaffected individuals (IV29, V-4, V-5, and V-6) were carriers of the linked haplotype (data not shown). Taken together, the clini-
59 Donor site
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GCCCAGgtaagcgcca AGCAAGgtgagtgcag AGAAAGgtaagggggc GCGGAGgtgagccatt GGGAAGgtgaacagga AAGCAGgtaggagagg GCCAGGgtgagccatc CAGCAGgtaagaccca AAGAGGgtgagcggct CCCAGTgtgagtgag GGCCAGgtgaggcacc CTGAGCgtaagtgtcc CAGCAGgtgtgtatgt CCTATGgtgagtgtcc AGAAAGgtaggtcctc CAGATGgtgaggaggc CACCAGgtgctttcca GCCCAGgtgagtggga
cal presentation and suggestive linkage to chromosome 8p12 indicated that hairless was a strong candidate gene to analyze for an underlying mutation. Mutation Identification To screen for a mutation in the human hairless gene, exons and splice junctions were PCR amplified from genomic DNA and sequenced directly. Sequence analysis of exon 3 of the hairless gene from two affected individuals (IV-2 and V-7) revealed a homozygous complex 22-bp out-of-frame deletion consisting of a 1-bp (delC) at nucleotide position 1256 and a 21-bp (del21) extending from nucleotide position 1261–1281. Together, the deletions led to a frameshift and premature termination codon 77 bp downstream within exon 3 (Fig. 4). The mutation, designated 1256delC;1261del21, was present in the heterozygous state in the obligate carriers in the family. DISCUSSION
FIG. 2. Transcript analysis of the human hairless gene. Expression of hairless mRNA was noted in all areas of the brain including A1, whole brain; A2, amygdala; A3, caudate nucleus; A4, cerebellum; A5, cerebral cortex; A6, frontal lobe; A7, hippocampus; A8, medulla oblongata; B1, occipital lobe; B2, putamen; B3, substantia nigra; B4, temporal lobe; B5, thalamus; B6, subthalamic nucleus; B7, spinal cord; and G1, fetal brain. Lower levels of expression were noted in C4, colon; C8, stomach; D7, salivary gland; E3, small intestine; F1, appendix; and F3, trachea.
The genomic organization of the hairless gene is highly conserved between human and mouse. In addition, the amino acid sequences of murine and primate hairless are 85 and 95% identical to the human homolog, respectively (Ahmad et al., 1998a; and unpublished results). Comparison of the intron– exon positions of the human hairless gene with those of the mouse homolog shows almost exact conservation of the positioning of the introns. The gene spans over 14 kb on human chromosome 8p12 and is encoded by 19 exons ranging from 70 to 793 bp in size (Fig. 1). Similarly, no significant differences in intron size were observed between the human and the mouse hairless genes. Hairless is a putative single zinc-finger transcription factor, with only two significant regions of
FIG. 3. Clinical and histological findings in the family with papular atrichia. (A) Pedigree of the family with obligate carriers indicated with a dotted figure, and affected individuals as solid figures. Double lines represent consanguineous unions, and the proband (individual V-7) is indicated by an arrow. (B) Clinical appearance of papular atrichia in individual V-7. The patient is nearly devoid of scalp hairs and has sparse eyebrows and eyelashes. (C) Histological findings reveal the complete absence of normal hair follicle structures, which are instead replaced by residual epithelial cell conglomerates and large dermal cysts. No excessive perifollicular infiltrate is visible, and the sebaceous glands, sweat glands, and interfollicular epidermis are normal (hematoxylin & eosin, 1253 magnification).
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FIG. 4. Sequence analysis of the hairless gene. (Top) The wildtype sequence of exon 3. (Middle) Sequence analysis of a heterozygous carrier. (Bottom) The 22-bp deletion in the homozygous state in an affected individual. The arrow and bar above the wildtype sequence in the top panel represent the sequence that is deleted in the homozygous state in the patient in the bottom panel.
homology to any known proteins. First, the six-cysteine zinc-finger motif contained within exon 6 of the human and mouse hairless genes (amino acids 600 – 625) shares weak homology with TSGA, a testis-specific putative transcription factor of unknown function (Hoog et al., 1991). Supporting evidence for the functional importance of the zinc-finger domain comes from mutational studies in an Irish family with atrichia, in which affected individuals are homozygous for a missense mutation between the fourth and the fifth cysteines of the zinc-finger (Ahmad et al., 1998b). The second region of weak homology is found toward the 39 end of the gene, in the region extending from amino acids 704 to 1031 of the human sequence. In this region, significant homology is found between hairless and a family of proteins known as TRIPs (thyroid hormone receptor interacting proteins) (Lee et al., 1995). The signature motif (LXXLL) for TRIPs and other transcriptional coactivators is necessary and sufficient to mediate binding of this class of proteins to liganded
nuclear receptors (Heery et al., 1997). It is noteworthy that hairless contains two LXXLL motifs, one in exon 5 (LCRLL, amino acids 566 –570) and a second within the region of TRIP homology (LCELL, amino acids 758 –762), adding evidence in support of functional significance of this region of the gene. Further implication of the region spanning from 704 to 1031 in functional integrity of hairless comes from mutation analysis in a Pakistani atrichia family with a missense mutation in the same region (Ahmad et al., 1998a). This rare form of inherited human hair loss was named atrichia with papular lesions in 1950 and was characterized by normal hair formation at birth followed by hair loss associated with the formation of comedones and follicular cysts (Fredrich, 1950). Later, in 1989, the human disease was first proposed as a homolog of the hairless mouse mutation (Sundberg et al., 1989). The molecular basis of the hairless mouse phenotype was shown to be the result of a murine leukemia proviral insertion into intron 6 of the hairless
HUMAN HAIRLESS GENE (HR) AND MUTATION IN CONGENITAL ATRICHIA
gene, resulting in aberrant splicing and a moderately severe phenotype (Cachon-Gonzalez et al., 1994). A second, phenotypically more severe allelic mutation, known as rhino, is the result of more deleterious nonsense and deletion mutations in the hairless gene (Ahmad et al., 1998c,d; Panteleyev et al., 1998a). Recently, the predicted connection between hairless mice and papular atrichia in humans was confirmed when mutations underlying this disorder were identified in the human hairless gene (Ahmad et al., 1998a,b; Zlotogorski et al., 1998). The mutation described in this study is the second reported deletion mutation in the human hairless gene. Since the mutation results in a frameshift and a downstream premature termination codon, we predict an absence of functional mRNA due to nonsense-mediated mRNA decay (Maquat, 1996) and consequently, an absence of hairless protein. Interestingly, however, in contrast to rhino and hairless mice, there appears to be no difference in phenotypic severity between human patients with different types of mutations in the hairless gene. It is intriguing that despite the high levels of hairless gene expression in the brain, neither rhino or hairless mice, nor any of the human atrichia patients we have studied, exhibit any detectable neurological phenotype or developmental delay reflecting aberrant hairless expression in developing CNS. The findings presented in this study extend the body of evidence implicating different types of mutations in the hairless gene as the underlying cause of congenital atrichia in humans. Previously, it was shown that hairless gene expression is primarily restricted to the skin and brain (Cachon-Gonzalez et al., 1994; Thompson, 1996; Ahmad et al., 1998a). To more fully characterize the sites of hairless expression, a dot blot containing poly(A) 1 RNA from several human tissues at different developmental stages was hybridized with the human hairless cDNA probe. In addition to high levels of gene expression detected in various parts of the brain, lower levels of hairless gene expression were also observed in colon, stomach, pituitary gland, salivary gland, small intestine, and appendix (Fig. 2). Although the precise function of hairless in the brain is still elusive, hairless protein has been shown to interact directly and specifically with thyroid hormone receptor, the same protein that induces its expression. Thus, in the brain, hairless appears to function both as a downstream target and as an upstream regulator of thyroid hormone action, potentially as a transcriptional corepressor (Thompson, 1996; Thompson and Bottcher, 1997). Further, a thyroid hormone response element (TRE) was identified 9 kb upstream of the transcriptional start site in the rat hairless gene. The TRE in the human and mouse hairless genes has not been identified as yet, and it remains to be determined whether hairless is similarly regulated by thyroid hormone in the skin. Interestingly, when hairless cDNA was amplified from human skin fibroblast total mRNA for generating the probe for the dot blot, we noted the
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consistent and complete absence of exon 17 from several independent RT-PCR products. This finding suggests that hairless may be subject to tissue-specific alternative splicing events, which may in turn specify differential cell-type specific functions. The cellular events leading to the development of hairlessness involve a premature and massive apoptosis in the hair matrix cells, together with a concomitant decline in Bcl-2 expression, a loss of NCAM positivity, and a disconnection with the overlying epithelial sheath essential for the movement of the dermal papilla (Panteleyev et al., 1998b,c,d). As a consequence, the dermal papilla remains stranded in the dermis, and indispensable messages between the dermal papilla and stem cells in the bulge are not transmitted, thus no further hair growth occurs. In hairless mice and in humans with congenital atrichia, we postulate that the absence of functional hairless protein leads to initiation of a premature and aberrant catagen phase due to abnormal apoptosis, dysregulation of cell adhesion, and defects in dermal papilla-derived signaling that normally control catagen-associated hair follicle remodeling (Panteleyev et al., 1998b,c,d). These observations suggest that a crucial role of the hairless protein may be involved in maintaining the balance between cell proliferation, differentiation, and apoptosis during hair follicle cycling. The identification of mutations in the hairless gene in humans and mice with congenital atrichia underscores a crucial role of the hairless gene product in the regulation of hair growth. Delineation of the genomic organization of the human hairless gene will not only facilitate the identification of mutations in patients with congenital atrichia, but also advance functional studies into the role of the hairless gene product in hair follicle morphogenesis, hair growth, and hair cycling. ACKNOWLEDGMENTS We appreciate the generous participation of the family members in this study. This work was supported in part by grants from the National Alopecia Areata Foundation (A.Z. and A.M.C.) and the NIH-NIAMS Skin Disease Research Center (P30-AR44535) in the Department of Dermatology at Columbia University.
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