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Identification of a novel splice site mutation in the human hairless gene underlying atrichia with papular lesions

European Journal of Dermatology. Volume 15, Number 5, 332-8, September-October 2005, Genes and skin

Summary

Author(s) : Mauro Paradisi, Maureen Massé, Amalia Martinez-Mir, HaMut Lam, Cristina Pedicelli, Angela M Christiano , Istituto Dermopatico Dell’Immacolata, Rome, Italy, Department of Dermatology, Columbia University, College of Physicians and Surgeons, 630 West 168th Street VC-1526, New York, NY 10032, USAFax: (+1) 212 305 7391., Department of Genetics and Development, Columbia University, New York, USA.

Summary : Atrichia with Papular Lesions (APL) is a rare autosomal recessive disorder characterized by complete hair loss that begins shortly after birth with the development of papular lesions on various regions of the body. Since the establishment of hairless (HR) gene mutations as the cause of this disorder, several patients previously assumed to suffer from alopecia universalis have been subsequently diagnosed with APL. In this study we have identified a novel splicing mutation, IVS8+2T→G, in the hairless gene. This mutation most likely abolishes normal splicing of exon 8 and potentially leads to out-of-frame skipping of this exon and a downstream premature termination codon (PTC). Our findings contribute to the growing body of HR mutations implicated in APL and provide further evidence for the differentiation of APL from alopecia universalis.

Keywords : alopecia, atrichia with papular lesions, autosomal recessive, hairless, premature termination codon, splice site mutation

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ARTICLE

Auteur(s) :, Mauro Paradisi¹, Maureen Massé², Amalia Martinez-Mir², HaMut Lam², Cristina Pedicelli¹, Angela M Christiano^2,3

¹Istituto Dermopatico Dell’Immacolata, Rome, Italy
²Department of Dermatology, Columbia University, College of Physicians and Surgeons, 630 West 168th Street VC-1526, New York, NY 10032, USAFax: (+1) 212 305 7391.
³Department of Genetics and Development, Columbia University, New York, USA

accepté le 18 Avril 2005

There are several forms of rare, inherited alopecias, including atrichia with papular lesions (APL) (OMIM #209500). APL is an autosomal recessive disorder characterized by complete hair loss that begins in infancy [1]. Normal hairs are usually present at birth, however during the first months of life, the neonatal hairs are irreversibly shed. Patients display a complete lack or near complete lack of scalp hair, sparse eyebrows and eyelashes, and an absence of secondary axillary, pubic, and body hair [2]. During the first few years of life, papules begin to appear that are distributed particularly under the midline of the eye, on the face, and on the extremities. Patients show no abnormalities of the teeth, nails, and sweating and their growth and development is unaffected [2]. On biopsy, mature hair follicles are absent and cysts containing cornified material are seen [2, 3]. Linkage analysis was used to map the APL locus to chromosome 8p12, a region containing the human hairless gene (HR) [3-5]. HR mutations have been detected in APL patients from many different backgrounds including, Arab Palestinian, Japanese, Mexican, Pakistani, and Polish [6]. To date, there have been 27 hairless mutations reported in the literature (table 1; [2, 4, 5, 7, 8, 18-29]). The majority of APL cases reported are the result of consanguineous unions, although recently, compound heterozygous cases have begun to emerge [7, 8]. APL cases are likely to be underreported however, since they are often mistaken for alopecia universalis, a much more common disease [6, 8]. It is thought that the misdiagnosis arises in part from the notion that APL results only from consanguineous unions and is rarely observed in clinical practice [8]. Furthermore, there has been a relative lack of awareness of APL, and until recently there have been no standard criteria for the diagnosis of APL [2]. In this study, we report a novel homozygous HR mutation, IVS8+2T→G, in two siblings affected with APL.

Materials and methods

( Table 1 )The proband was a 20 year-old male of Italian descent. At birth, he presented with alopecia of the scalp and body, with the exception of sparse hairs in the occipital and apical areas of the scalp, which were shed after a few months. At approximately 6 years of age, the patient developed multiple hyperkeratotic, whitish follicular papules, initially localized on the arms, and during subsequent years they progressed to the thighs, scalp, and lateral region of the face. Skin biopsies of the scalp, as well as a papular lesion, were performed and histological examination was consistent with a diagnosis of APL ( (figure 1C ) and D). The patient used topical steroid treatments for many years without benefit. Currently, he has complete absence of hair on the scalp, eyebrows, eyelashes, axilla, and body ( (figure 1A ) and B). A 24 year-old sister of the proband also presented with similar symptoms and has had a similar disease progression. The patient’s parents and his two other siblings are unaffected. Although the parents report no history of consanguinity, they originate from two nearby towns in Italy.

To identify the mutation responsible for APL in the proband, all exons and splice junctions of the HR gene were PCR amplified from genomic DNA and sequenced directly in an ABI Prism 310 Automated Sequencer, using the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems, Foster City, CA), following purification with Centriflex Gel Filtration Cartridges (Edge Biosystems, Gaithersburg, MD). The mutations were confirmed using restriction endonuclease digestion.
Table 1 APL Mutations

Nonsense mutations
R33X	Exon 2	Mediterranean	[18]	Zlotogorski et al. 2002b
R154X	Exon 2	South Korean		Ashoor et al., 2005 (submitted)
Q260X	Exon 3	Caucasian		Michailidis et al. 2005 (submitted)
Q478X	Exon 4	Pakistani	[19]	Sprecher et al. 1999b
Q515X	Exon 4	South Korean		Ashoor et al., 2005 (submitted)
R613X	Exon 6	Japanese	[20]	Ahmad et al. 1999b
W699X	Exon 8	Caucasian		Michailidis et al. 2005 (submitted)
Q1176X	Exon 19	German	[21]	Henn et al. 2002
Missense mutations
E583V	Exon 5	Italian	[22]	Paradisi et al. 2003
C622G	Exon 6	Polish	[23]	Aita et al. 2000
N970S	Exon 14	South Tyrolian	[24]	Kruse et al. 1999
D1012N	Exon 15	Arab Israeli	[25]	Djabali et al. 2004
T1022A	Exon 15	Pakistani	[4]	Ahmad et al. 1998
V1056M	Exon 16	Arab Palestinian	[2]	Zlotogorski et al. 2002a
V1136D	Exon 18	Pakistani	[5]	Cichon et al. 1998
Deletions/insertions
177del11	Exon 2	Jewish Israeli	[26]	Zlotogorski et al. 2003
189-199del	Exon 2	Jewish Moroccan	[7]	Indelman et al. 2003
1256delC;1261del21	Exon 3	Arab Palestinian	[27]	Ahmad et al. 1999a
2001delCCAG	Exon 7	Mexican	[24]	Kruse et al. 1999
2147delC	Exon 9	Arab Palestinian	[28]	Zlotogorski et al. 1998
2847-2delAG	Exon 14	German	[21]	Henn et al. 2002
3434delC	Exon 18	Arab Israeli	[29]	Sprecher et al. 1999a
Splice site mutations
1557-1 G→T	Intron 4	Iraqi	[8]	Paller et al. 2003
IVS8+2T→G	Intron 8	Italian		This study
2776+1 G→A	Intron 12	Omani	[5]	Cichon et al. 1998
2776+2 insT	Intron 12	Australian	[8]	Paller et al. 2003
2847-3 C→G	Intron 13	Russian	[8]	Paller et al. 2003

Results

Sequencing of the PCR product corresponding to exon 8 revealed a homozygous splicing mutation, IVS8+2T→G in the affected individuals ( (figure 2) ). The parents and the unaffected siblings are heterozygous carriers of the mutation ( (figure 2) ). Mismatched PCR was performed using a mismatched reverse primer (5’-GGTGACATGCCCTGGGTCGT-3’) which introduces an RsaI restriction site in the presence of the wild-type sequence. The mutation IVS8+2T→G abolishes this restriction site. Enzymatic restriction of the mismatched PCR product was used to confirm which individuals were carriers of the mutation. The mutation was detected in all affected individuals in the homozygous state and in the heterozygous state in the parents and the unaffected siblings, confirming the sequencing findings ( (figure 2) ).

Discussion

There are numerous disorders of congenital hair loss (table 2)( Table 2 ), [2, 9, 30-49]. APL can be differentiated from these disorders through family history, clinically and via histopathologic and genetic analyses. A family history delineating a pattern of autosomal recessive inheritance and possibly consanguinity are distinguishing features of APL. Clinically, the atrichia of APL is discernible by the presence of hair at birth which is shed irreversibly in the first few months of life. In rare cases, patients are born without hair and never grow hair [2]. Furthermore, papules found on various regions of the body, particularly under the midline of the eye, on the face, and on the extremities, are a distinctive feature of APL. Histologically, the absence of mature hair follicles and the presence of dermal cysts, further differentiate APL from other forms of congenital hair loss. Finally, a crucial distinction is the identification of mutations in the HR gene, a finding that has not been demonstrated in any of the other disorders of congenital hair loss. APL can also be distinguished from other congenital hair loss disorders by the absence of extracutaneous abnormalities which are found in other diseases in the differential such as Netherton’s syndrome (NS), Naxos disease, human nude, Menkes’ disease (MK), the ectodermal dysplasias, and juvenile macular dystrophy and congenital hypotrichosis (HJMD) [32, 33, 36-39, 43-49]. The pattern of hair loss in APL is also distinctly different from many of the other disorders of congenital hair loss such as congenital triangular alopecia, NS, localized autosomal recessive hypotrichosis, Marie Unna Hypotrichosis (MUHH), hypotrichosis simplex, and HJMD [30, 32-35, 40-42, 48, 49]. Notable hair textural differences exist as well, such as those present in NS, Naxos disease, MK, and MUHH [32, 33, 36, 37, 39-41].

A disorder deserving particular mention is hereditary vitamin D resistant rickets (HVDRR). The clinical presentation of hair loss and papular lesions are nearly identical in APL and HVDRR. Moreover, the findings on histological examination can also appear similar in these two disorders, both manifesting with an absence of mature hair follicles and the presence of dermal cysts [9]. HVDRR, however, has two major features that have not been demonstrated in APL patients: mutations in the vitamin D receptor gene (VDR) and the presence of rickets. The clinical and pathological similarities between APL and HVDRR suggest that VDR and HR, which are both zinc-finger proteins, may reside in the same genetic pathway that controls postnatal hair cycling [9].

The HR gene is highly expressed in the brain and skin [4]. The protein product of this gene, also termed hairless (HR), has been shown to contain a single zinc-finger domain and is a putative transcription factor [10]. HR is thought to function as an essential regulator of apoptosis during normal hair follicle regression (catagen) and appears to function in the cellular transition to the first hair cycle. In the absence of HR, hair follicles disintegrate and a new hair cannot be formed [11-13]. This hypothesis correlates with the phenotype observed in APL patients, and further supports the implication of HR mutations in the pathogenesis of APL.

In our patient, one possible consequence of the HR mutation IVS8+2T→G (which likely abolishes normal splicing of exon 8) is the out-of-frame skipping of this exon. Exon skipping would result in a premature termination codon (PTC) downstream in exon 10 (nucleotide position 3701, Gen Bank Access. No. NM_005144), generating an mRNA that would likely be degraded by nonsense mediated mRNA decay (NMRD) [14]. Exon skipping is a well-known potential consequence of mutations that disrupt consensus splicing sequences [15, 16]. The presence of exon skipping in this case could not be assessed however, since mRNA samples from the proband and his affected sibling were not available. We have calculated the splicing efficiency score that results from the presence of this mutation [17]. The mutation IVS8+2T→G is predicted to abolish normal splicing of exon 8 of both alleles since the invariant GT immediately following exon 8 is converted to GG. The score for the wild-type sequence is 94.34 and that for the mutant sequence is 76.09, suggesting a significant decrease in splicing efficiency as a result of the mutation.

In summary, we report a novel HR mutation responsible for APL in two siblings. These findings extend the body of evidence for HR mutations implicated in APL. As additional HR mutation-induced cases of APL emerge, the prevalence of this disorder can be more adequately assessed and awareness of its potentially higher prevalence can lead to the consideration of APL in cases possibly misdiagnosed as alopecia universalis. APL patients can thereby be spared unnecessary and invariably ineffective treatment for alopecia universalis and accurate genetic counseling can be offered, as the allelic series of HR mutations continues to expand.
Table 2 Differential diagnosis of congenital hair loss

Disease	Mutated Gene/Mode of inheritance	Distinguishing features	Ref.	Reference
Atrichia with papular lesions (APL)	Hairless (HR) Autosomal recessive	Hair loss present at birth or irreversible loss of hair after several months; Complete lack or nearly complete lack of scalp hair; sparse eyebrows and eyelashes; lack of secondary axillary, pubic, or body hair; cutaneous papules; dermal cysts on biopsy	[2]	Zlotogorski et al., 2002
Hereditary vitamin D resistant rickets (HVDRR)	Vitamin D receptor (VDR) Autosomal recessive	Hair generally present at birth but is lost within 12 months; dermal cysts on biopsy; rickets	[9]	Miller et al., 2001
Congenital triangular alopecia (CTA)	Gene unknown; Possible paradominant inheritance	Bald patch in temporal region with approximately triangular shape; usually unilateral	[30]	Happle, 2003
Monilethrix	Type II hair keratin genes (hHb1, hHb3, hHb6) Autosomal dominant	Periodic beading of hair shaft, hair fragility, scarring alopecia	[31]	van Steensel et al., 2005
Netherton’s syndrome (NS)	SPINK5 Autosomal recessive	Sparse, brittle hair; bamboo hair; congenital ichthyosis; atopic manifestations; failure to thrive	[32, 33]	Chavanas et al., 2000a; Chavanas et al., 2000b
Localized autosomal recessive hypotrichosis (LAH)	Desmoglein 4 (DSG4) Autosomal recessive	Hypotrichosis affecting the scalp, trunk, and extremities, and largely sparing the facial, pubic, and axillary hair; sparse, fragile, broken scalp hairs with a characteristic appearance; histologically hair follicles are abnormal and hair shafts are thin and atrophic and often appear coiled up within the skin	[34, 35]	Kljuic et al., 2003; Moss et al., 2004
Naxos disease	Desmoplakin (DSP) and Plakoglobin (JUP) Autosomal recessive	Wooly, sparse hair; keratoderma; cardiomyopathy	[36, 37]	Mckoy et al., 2000; Norgett et al., 2000
Human nude	Winged helix nude (WHN) Autosomal recessive	Complete absence of scalp hair, eyebrows, and eyelashes; dystrophic nails; severe immunodeficiency	[38]	Frank et al., 1999
Menkes’ disease (MK)	ATP7a X-linked recessive	Sparse, depigmented and lusterless hair; trichorrhexis nodosa and pili torti; cutis laxa; progressive neurological abnormalities; bony changes; GI hemorrhage and diarrhea	[39]	Kodama et al., 2001
Marie Unna Hypotrichosis (MUHH)	Gene unknown; Maps to chromosome 8p21.3 Autosomal dominant	Sparse scalp hair at birth; hair growth during childhood, but hairs are coarse and wiry; hair loss accelerates in the years close to the onset of puberty; hair is lost in a Norwood (or Hamiltonian) pattern; all affected persons have little or no body hair, eyelashes, eyebrows or secondary sexual hair	[40, 41]	van Steensel et al., 1999; He et al., 2004
Hypotrichosis simplex (HTSS)	Corneodesmosin (CDSN) Autosomal dominant	Normal hair in early childhood but progressive scalp hair loss begins in the middle of the first decade and by the third decade nearly complete baldness occurs; body hair, beard, eyebrows, axillary hair, teeth, and nails are normal	[42]	Levy-Nissenbaum et al., 2003
X-linked hypohidrotic ectodermal dysplasia (ED1)	Ectodysplasin (EDA) X-linked recessive	Sparse hair, abnormal teeth, and decreased sweating	[43]	Yotsumoto et al., 1998
Autosomal hypohidrotic ectodermal dysplasia	Ectodysplasin anhidrotic receptor (EDAR) Autosomal dominant and Autosomal recessive forms	Sparse hair, abnormal teeth and decreased sweating	[44, 45]	Monreal et al., 1999; Ho et al., 1998
Hypohidrotic ectodermal dysplasia with immune deficiency (HED-ID)	IKK-gamma (NEMO) X-linked recessive disorder	Normal or fine, sparse scalp hair; dysgammaglobulinemia and recurrent infections	[46]	Zonana et al., 2000
Ectodermal dysplasia/skin fragility syndrome	Plakophilin 1 (PKP1) Autosomal recessive	Sparse scalp hair, trauma-induced skin fragility, nail dystrophy, decreased sweating	[47]	McGrath et al., 1997
Juvenile macular dystrophy and congenital hypotrichosis (HJMD)	P-cadherin (CDH3) Autosomal recessive	Born with seemingly normal hair but develop alopecia of the scalp after a few months; during puberty, partial regrowth of short and sparse hair can occur; progressive macular degeneration likely leading to blindness during the second to third decade of life	[48, 49]	Sprecher et al., 2001; Indelman et al., 2002

Acknowledgements

The authors would like to thank the family members for their contribution to this study. This work was supported by a grant from the NIH MIAMS USPHS grant RO1 AR 47338 (AMC), and T32 AR07605 (MM).

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