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Atrichia with papular lesions (APL) (OMIM#209500) is a rare autosomal recessively inherited form of irreversible alopecia characterized by papular lesions of keratin-filled cysts on various regions of the body. Males and females are equally affected and present with a distinct pattern of total hair loss on scalp, axilla and body. It begins shortly after birth with the development of hair loss, and patients are normally devoid of eyelashes and eyebrows. Mutations in the hairless (HR) gene have been previously shown to be responsible for APL.
In this study, we studied the molecular basis of APL in three unrelated families of Pakistani origin.
Molecular analysis of the HR genes was performed on genomic DNA from probands and family members.
DNA sequencing of the HR gene in family A revealed a novel homozygous 2 bp deletion in exon 6 leading to a frameshift and a downstream premature termination codon in exon 8 (1782-83delAG). In family B, we identified a novel homozygous deletion of a G nucleotide at the exon 15–intron 15 boundary, termed 3097delG. Family C carries a previously reported missense mutation consisting of an A-to-G transition at nucleotide 276 resulting in the mutation N970S in exon 14.
Two mutations identified in this study are novel mutations in the HR gene and extend the body of evidence implicating the hairless gene family in the pathogenesis of human skin disorders. The one previously reported mutation suggests it may represent a recurrent mutation, or alternatively, an allele that is widely dispersed around the world.
To date, the genetic basis of several forms of congenital hair loss has been identified, many of which are inherited in an autosomal recessive manner . One example is atrichia with papular lesions (APL) (OMIM#209500), characterized by complete hair loss which begins shortly after birth. The scalp, axillae and the entire body become completely devoid of hairs, whereas the eyelashes and eyebrows may be just partially affected . This form of hair loss is irreversible and histology confirms an absence of mature hair follicles. After approximately 2 years of age, affected individuals begin to develop papular lesions of keratin-filled cysts on various regions of the body, which represents a unique cutaneaous finding in APL among inherited alopecias. Other ectodermal structures such as nails, sweat glands or teeth are not affected [3,4].
The human homolog of the hairless gene has been identified and mapped to human chromosome 8p12, a region syntenic to mouse chromosome 14 [5–7]. The hairless gene product is a transcriptional co-regulator with a single zinc-finger domain, which is highly expressed in the skin and the brain [7–9]. Furthermore, mutations in the hairless (HR) gene were identified in many APL families and to date, more than 30 hairless mutations have been reported [10–18]. Although APL could potentially be diagnosed by its clinical features alone, or by skin biopsy, APL remains frequently misdiagnosed as the more common disorder, alopecia universalis (AU). This is maybe due to a lack of awareness, and/or the notion that APL patients are rare and found exclusively in consanguineous families. Therefore, many more mutations are likely to be discovered, since APL appears to be more common than previously considered . To date, HR mutations have been found in APL patients from various ethnic backgrounds, including Pakistani, Mediterranean, Arab Palestinian, Caucasians, Japanese and Polish [10–18]. In this study, we identified three Pakistani families originating from different regions of Pakistan with clinical manifestations of APL and high degree of consanguinity. All affected individuals were identified by generalized scalp and body alopecia, sparse eyebrows and lashes as well as papules (Fig. 1A). Direct DNA sequencing of the HR gene in APL patients identified unique HR mutations in three families of Pakistani origin.
Genomic DNA was isolated from blood following informed consent using the Pure-Gene DNA Isolation Kit (Gentra Systems) and PCR was performed using HR specific primers to amplify the exons of HR as previously described . Briefly, PCR products were purified using Rapid PCR Purification Systems (Marligen Biosciences) and eluted in H2O. Sequencing PCR was performed using purified fragments and either forward or reverse primers (10 pmol) with BigDye® Terminator v3.1 Cycle Sequencing Kits (ABI). Samples were purified using Centriflex Gel Filtration Cartridges (Edge Biosystems), resuspended in Hi Dye.
In order to analyze the homozygous deletion G at the intron/exon boundary 15, designated 3097delG in family B, mismatch allele-specific PCR was performed using a forward primer 5′-GACACACCACTGCCTGCCTGGCACCGGCCTCAGAAA-3′, and a reverse primer 5′-CTGAGGAGGAAAGAGCGCTC-3′. Note that the G > C and A >T substitutions were introduced into the forward primer to generate the EcoNI restriction enzyme site only in the PCR product from the wild-type allele (shown in bold and underlined). The amplification conditions were 94 °C for 2 min, followed by 35 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 30 s, with a final extension at 72 °C for 7 min. The PCR products were digested by the EcoNI at 37 °C for overnight, which were analyzed on 1.5% agarose gels.
To confirm the missense mutation, direct PCR was used. The DNA mutation N970S was confirmed as a mutation by digestion of the corresponding PCR-amplified product with DdeI (New England Biolabs, Beverly, MA). Fifty unrelated, unaffected control DNAs from Pakistani individuals were checked for the mutation N970S by PCR, digestion and run on a 3% agarose gel and visualized by ethidium bromide staining.
In this study, we identified mutations in affected individuals in three families of Pakistani origin. To screen for mutations in the HR gene, all exons and splice junctions were PCR amplified and sequenced directly. Sequence analysis of each exon of the hairless gene in the proband of family A revealed a homozygous 2 bp deletion in exon 6 (Fig. 1). The father was a heterozygous carrier. The mutation was designated 1782delAG, and results in a frameshift and a premature termination codon 230 bp downstream in exon 8. All affected members from family B show a homozygous deletion G at the intron/exon boundary 15, termed 3097delG (Fig. 2). This mutation most likely abolished normal splicing of exon 15, and leads to a frameshift and premature stop codon 214 bp downstream in exon 17. The mismatch allele specific PCR showed that 50 unrelated healthy control individuals (100 chromosomes) from Pakistan do not carry the mutation (Fig. 2).
The two affected individuals of family C (Fig. 3) carried a missense mutation consisting of an A-to-G transition (AAC to AGC) resulting in the substitution N (asparagine) to S (serine) at amino acid 970 in exon 14, and is designated N970S. The mutation creates a restriction endonuclease site for the enzyme DdeI in exon 14, resulting in a cleavage into products of 145 and 151 bp. The wild-type PCR product contains no restriction site for the enzyme, resulting in a band of 296 bp. The carrier individuals display the 296 bp band together with the superimposed 145 and 151 bp bands, indicative of heterozygosity for the mutant allele.
Since so many disorders of congenital hair loss exist, it is important to establish the correct diagnosis in order to avoid unnecessary and ineffective treatment options. Since APL is resistant to any treatment modalities due to destruction of the hair follicle, a careful family history, clinical exams and genetic analysis in the hairless gene (HR) on chromosome 8p21 would help to establish a definitive diagnosis.
Although cases from small non-consanguineous families have been reported , consanguinity and autosomal recessive inheritance still remains a distinguishing feature of APL. Interestingly, there is no genotype–phenotype connections in APL patients. The various HR mutations that have been discovered in APL patients, in this report as well as previous reports, do not indicate a correlation between the type of mutation, its location and the severity of the phenotype. All patients with APL are affected to the same degree of clinical serverity, irrespective of the nature of the mutation.
One possible consequence of the mutation 3097delG (which likely abolished normal splicing of exon 15) in the patient of family B is an out-of-frame skipping of exon 15. This mutation results in a deletion of G of the receptor splice side of exon 15 (3097delG). The wild-type sequence of this splice site is: (exon 15) … GCACAGAAAG/gtaggtcctcggcca … (intron 15). The mutation results in creation of the mutant splice junction: (exon 15) GCACAGAAAG/taggtcctcggcc (intron 15), or alternatively (exon 15) GCACAGAAA/gtaggtcctcggcc (intron 15), in which the consensus G is deleted either within the exon, or within the gt of the donor splice site. The consequence of this mutation could result in the production of several mutant mRNA species. Exon skipping is a well-known potential consequence of mutations which disrupt consensus splicing sequences [18–20]. The presence of exon skipping in this case could not be assessed as tissue samples for analysis of mRNA species generated by this mutation were not available for study, however, exon 15 is 119 bp in size, predicting its skipping would be out-of-frame. The other possibility could be splicing after the AAA at the end of the exon, creating a 1 bp deletion, and therefore generating a frameshift mutation.
N970 is a conserved amino acid in the HR sequence compared among mouse, rat, human and monkey. Interestingly, this mutation has previously been reported in a South Tyrolian APL family [16,21], suggesting it may represent a recurrent mutation, or alternatively represents an allele that is widely dispersed around the world. The recurrent missense mutation reported in exon 14 of the family C and the previously reported South Tyrolian family suggests the C-terminus, including the second TR-ID and the jumonji C domain, to be critical regions for HR function. The C-terminus of HR is a highly conserved region among mouse, rat, monkey and human protein sequences. It is possible that alterations in or loss of this highly conserved region of the HR protein results in HR inactivation, which suggests a critical domain to HR function, resides in this region. The C-terminus of hairless contains a highly conserved region related to the JmjC domain. Five mutations with impaired activity have been reported to be located in this JmjC domain (N970S, D1012N, V1056M and V1136D) showing that these mutations disrupt binding to the vitatmin D receptor .
Furthermore, the fact that this mutation was present in this previously reported patient of South Tyrolian origin and in our patient, suggest that they may share a common founder allele.
By expanding the database of allelic series of HR mutations (Fig. 4), diagnosis of APL becomes increasingly accurate, and the invariably ineffective treatment of APL patients can be minimized. A refined clinical history, including autosomal recessive inheritance and the findings of small to large numbers of papules distributed over some or all of the following areas: scalp, cheeks, arms, elbows, buttocks, thighs and knees, and under the midline of the eye, as well as a scalp biopsy with the presence of dermal cysts, and in particular the molecular diagnosis of mutations in the HR gene, will help to make a definitive diagnosis of APL. Patients will benefit from genetic analysis as in many cases the diagnosis is done in retrospect, following the lack of response to any treatment modality over several years.
We are especially grateful to the patients and their families for their interest in this study. We highly appreciate the skillful help and excellent technical assistance of Ha Mut Lam. This study was supported in part by USPHS NIH grant from NIH/NIAMS R01 AR47338 (to AMC).