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Molecular and clinical characterization in Japanese and Korean patients with Hailey–Hailey disease: Six new mutations in the ATP2C1 gene
Journal of Dermatological Science (2008) 51, 31—36
www.intl.elsevierhealth.com/journals/jods
Molecular and clinical characterization in Japanese and Korean patients with Hailey—Hailey disease: Six new mutations in the ATP2C1 gene Takahiro Hamada a,*, Shunpei Fukuda a, Sachiko Sakaguchi a, Shinichiro Yasumoto a, Soo-Chan Kim b, Takashi Hashimoto a a
Department of Dermatology, Kurume University School of Medicine, Kurume, Japan Department of Dermatology and Cutaneous Biology Research Institute, Yonsei University College of Medicine, Yongdong Severance Hospital, Seoul, Republic of Korea
b
Received 10 January 2008; received in revised form 4 February 2008; accepted 11 February 2008
KEYWORDS Familial benign chronic pemphigus; Acantholysis; P-type ATPase; Ca2+; M2 helix
Summary Background: The autosomal dominant disorder Hailey—Hailey disease (HHD) results from mutations in the ATP2C1 gene, which encodes the human secretory pathway Ca2+/Mn2+-ATPase protein 1. To date, over 90 pathological mutations scattered throughout ATP2C1 have been described with no indication of mutational hotspots or clustering of mutations. No paradigm for genotype—phenotype correlation has emerged. Objectives: To determine the pathogenic ATP2C1 abnormality in additional patients with HHD in order to provide further contributions to the understanding of the molecular basis of this disorder and to add the data to the known mutation database. Methods: In this study, we investigated eight unrelated Japanese and Korean patients with HHD. We performed direct nucleotide sequencing of the ATP2C1 gene in all patients and RT-PCR analysis, using RNA extracted from a skin biopsy, in a patient with the mildest clinical features. Results: We identified seven different heterozygous mutations in seven of the eight investigated patients, including three new single nucleotide deletion/duplication mutations: c.520delC; c.681dupA; c.956delC, three new donor splice site mutations: c.360 + 1G > C; c.899 + 1G > T; c.1570 + 2T > C, as well as a previously described nonsense mutation: p.Arg153X. RT-PCR analysis in the mildest affected patient with a heterozygous c.360 + 1G > C mutation, demonstrated expression of a short in-frame mutant transcript with exon 5 skipping, which may account for the mild phenotype.
* Corresponding author. Tel.: +81 942 31 7571; fax: +81 942 34 2620. E-mail address: [email protected] (T. Hamada). 0923-1811/$30.00 # 2008 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2008.02.003
32
T. Hamada et al. Conclusions: The results expand the known mutation spectrum in HHD and show the importance of RNA analysis for understanding the genotype—phenotype correlations more precisely. # 2008 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction Hailey—Hailey disease (HHD; MIM 169600) is a rare autosomal dominant disorder characterized by abnormal keratinocyte adhesion in the suprabasal layers of the epidermis (acantholysis). The disorder, which usually presents in the third or fourth decade, demonstrates recurrent vesicular lesions, crusted erosions, and warty papules mainly on the neck, axillae, groin, and perineum. Lesions are induced or exacerbated by external factors such as sweating, frictions, and cutaneous infections. Ultrastructural studies of acantholytic cells in HHD reveal perinuclear aggregates of keratin intermediate filaments that have retracted from desmosomal plaques. Darier disease (DD; MIM 124200) is a similarly inherited skin disorder, which has clinical and histological overlap with HHD. DD was shown to be caused by mutations in the ATP2A2 gene on 12q24.1, which encodes the sacro/endoplasmic reticulum Ca2+ ATPase isoform 2 (SERCA2) [1]. Shortly after that report appeared, the molecular basis of HHD was shown to result from mutations in the ATP2C1 gene on 3q22.1, encoding the human secretory pathway Ca2+/Mn2+-ATPase protein 1 (hSPCA1) [2,3]. These proteins belong to the P-type ATPase superfamily, which is defined by the highly conserved phosphorylation sequence DKTGT [4]. No evidence of genetic heterogeneity in HHD has been found thus far. Over 90 pathological mutations scattered throughout the ATP2C1 gene have been described with no indication of mutational hotspots or clustering of mutations [2,3,5—19]. The hSPCA1 is located in the Golgi apparatus in human keratinocytes and serves to actively pump Ca2+ out of the cytoplasm [20]. These findings suggest that intracellular Ca2+ stores play an important role in regulation of epidermal cell—cell adhesion and differentiation. In this study, we investigated the ATP2C1 gene pathology in eight additional unrelated Japanese and Korean patients with HHD in order to provide further contributions to the understanding of the molecular basis of this disorder. We identified seven different mutations in seven of eight investigated familial or sporadic cases. Six of them were new mutations. Furthermore, we performed reverse transcription-polymerase chain reaction (RT-PCR) analysis in a patient with the mildest clinical fea-
tures to assess genotype—phenotype correlation in an ATP2C1 mutation resulting in aberrant splicing.
2. Materials and methods 2.1. PCR amplification of genomic DNA and mutation detection All described studies were performed following the guidelines of the medical ethical committee of Kurume University School of Medicine. Written informed consent was obtained from each individual, and the study was conducted according to the Declaration of Helsinki Principles. Genomic DNA from all individuals was extracted from peripheral blood samples using standard methods. For mutation analysis, PCR fragments were amplified with 28 pairs of primers designed for all exons of the ATP2C1 gene with upstream and downstream primers extending at least 50 bp into flanking introns. The mutation detection strategy consisted of heteroduplex scanning by denaturing gradient gel electrophoresis [21]. The corresponding PCR products showing heteroduplexes were then sequenced directly using Big Dye labeling in an ABI310 genetic analyser (Applied Biosystems, Foster City, USA). Potential mutations were confirmed by restriction endonuclease digestion or bi-directional sequencing and assessed by examining 100 control DNA samples, selected to match the patients’ ethnic backgrounds.
2.2. Genotyping Genotyping was performed by PCR amplification of genomic DNA using the fluorescent microsatellite markers D3S3514, D3S1596, D3S1587, and D3S1292, as described previously [3]. PCR products was then analysed on an ABI310 genetic analyser using Genescan 3.1.2 and Genotyper 2.5.2 software (Applied Biosystems).
2.3. RNA extraction and RT-PCR analysis RNA was only available from patient 1 in this study. Total RNA was isolated from a lesional groin skin sample using a commercial extraction kit (RNeasy Mini Kit, Qiagen K.K., Tokyo, Japan). RT was performed using another commercial kit (SuperScriptTM
Six new ATP2C1 mutations in Hailey—Hailey disease
33
Table 1 Clinical characterization in patients with HHD in this study Patients
Nationality
Sex
Age
Inheritance
Age at onset a
Affected skin lesion(s)
Severity
1 2 3 4 5 6 7
Japanese Japanese Japanese Korean Japanese Japanese Korean
M M M F M M F
57 42 47 62 44 58 55
Sporadic Sporadic Sporadic Familial Familial Sporadic Sporadic
50 20 30 40 20 40 20
Mild Severe Severe Moderate Moderate Moderate Severe
8
Japanese
F
52
Sporadic
40
Groin Axillae, back, and groin Axillae, groin, and perineum Axillae and groin Axillae, groin, and popliteus Axillae, groin, and perineum Neck, axillae, chest, groin, and cubital area Neck, axillae, cubital area, groin, and popliteus
a
Severe
Age at onset, 20, 10—20 years; 30, 30—40 years; 40, 40—50 years; 50, 50—60 years of age.
III First-Strand Synthesis System for RT-PCR, Invitrogen, Carlsbad, CA, USA). A pair of primers for RT-PCR extending across the site of the heterozygous c.360 + 1G > C mutation comprised: forward primer 50 -GCATCAGTTTGATGATGCCG-30 (GenBank NM_014382) and reverse primer 50 -GCGTAAGTCAGCAGGAACTC-30 . The RT-PCR products were subcloned into a PCR-compatible cloning vector (TOPO TA Cloning Kit, Invitrogen) and sequenced directly using Big Dye labeling in an ABI 310 genetic analyser (Applied Biosystems).
Table 2. Patients 3, 4, and 6 were heterozygous for single nucleotide deletion/duplication mutations (c.520delC, c.681dupA, c.956delC), which lead to frameshift and downstream premature termination codons (PTCs). Patients 1, 5, and 7 were heterozygous for donor splice site mutations (c.360 + 1G > C, c.899 + 1G > T, c.1570 + 2T > C) (Fig. 1b). Patient 2 was heterozygous for a nonsense mutation (p.Arg153X). Six of these mutations have not been reported previously. None of the mutations were found in 100 ethnically matched control DNA samples.
3. Results
3.3. RT-PCR analysis in the mild phenotype case reveals an aberrant mutant transcript with in-frame exon 5 skipping
3.1. Clinical features Two familial and six sporadic HHD cases were included in this study. In each patient, diagnosis was established by dermatologists on the basis of clinical and histopathological features. In the two familial cases, we only examined each proband. The patient profiles are summarized in Table 1. Patients 4 and 7 were of Korean origin, and the others were of Japanese origin. The ages at onset were distributed between 20 and 50 years. All patients except patient 1 demonstrated typical clinical features including scaly erythematous plaques, vesiculopustules, and painful erosions on the neck, axillae, chest, cubital area, groin, perineum, and popliteus. Patient 1 showed dusky erythema and papules with tiny erosions restricted to the groin (Fig. 1a). There were no skin abnormalities in other intertrigenous areas.
The mildest clinical manifestations were noted in patient 1, who was heterozygous for the mutation c.360 + 1G > C in intron 5. In cDNA from skin of patient 1, RT-PCR across the site of the c.360 + 1G > C mutation identified two bands of 232 and 196 bp compared with a single 232-bp band in the normal control sample (Fig. 1c). Subcloning and direct sequencing disclosed that the 196-bp band was an aberrant mutant transcript with inframe exon 5 skipping (Fig. 1c). The 232-bp band was the normal wild type transcript. These findings demonstrate the expression of the short in-frame mutant transcript with exon 5 skipping through the c.360 + 1G > C mutation, which may account for the mild phenotype.
3.2. Delineation of pathogenic ATP2C1 mutations in HHD
4. Discussion
Sequencing of genomic DNA in eight unrelated patients with HHD disclosed pathogenic mutations in each patient except patient 8, as detailed in
Among the P-type ATPase superfamily members, SERCA1 has been structurally and functionally well presented. The SERCA1 comprises three cytoplasmic
34
T. Hamada et al.
Fig. 1 Clinical features and molecular basis of patient 1. (a) Dusky erythema and papules with tiny erosions restricted to the groin. (b) Direct sequencing of genomic DNA of ATP2C1 shows a heterozygous G > C transition at the + 1 position of the exon 5 donor splice site, designated c.360 + 1G > C. (c) RT-PCR, subcloning and direct sequencing across the site of the c.360 + 1G > C mutation reveals a mutant transcript with in-frame exon 5 skipping (196 bp), as well as the normal transcript (232 bp).
domains, designated as A (actuator), N (nucleotidebinding), and P (phosphorylation) domains, and ten (M1—10) transmembrane helices [22]. Two high-affinity Ca2+-binding sites are located side-by-side within the transmembrane region, formed by M4— 6 and M8 helices. Both hSPCA1 and SERCA2 are thought to have similar structures and functions as SERCA1. Six crystal structures are now known that represent different states of the reaction cycle of ATPase [22—28]. For example, during the reaction cycle the cytoplasmic domains form a compact headpiece, then the A domain is tilted by 308 around an axis approximately parallel to the membrane, while the ATPase is phosphorylated [27]. Because the A domain is directly linked to M1, M2, and M3 helices, its movement inevitably causes rearrangement of these helices and leads to opening
of the lumenal gate and releasing the bound Ca2+ into the lumen. A number of mutations scattered throughout the ATP2C1 gene have been described. About 70% of them are nonsense, insertion/deletion or splice site mutations [7]. In this study, we have identified six new heterozygous frameshift or splice site mutations in the ATP2C1 gene in patients with HHD, as detailed in Table 2. The utility of computational tools that can accurately predict cryptic splicing, such as Splice Site Prediction by Neural Network (http://www.fruitfly.org/seq_tools/splice.html), has been described recently [29]. An analysis using this tool revealed that the c.1570 + 2T > C mutation resulted in a loss of a donor splice site, predicting out-of-frame exon 17 skipping; and the c.899 + 1G > T mutation resulted in a novel cryptic donor splice site 5-bp
Table 2 ATP2C1 mutations in patients with HHD in this study Patients
Mutation
Location
Putative protein domain a
Mutation in mRNA protein level b
1 2 3 4 5
c.360 + 1G > C p.Arg153X c.520delC c.681dupA c.899 + 1G > T
intron 5 exon 7 exon 7 exon 8 intron 11
M2 Actuator Actuator Actuator M4
6 7
c.956delC c.1570 + 2T > C
exon 12 intron 17
M4 Nucleotide
In-frame exon 5 skipping (R) PTC in exon 7 PTC in exon 8 PTC in exon 9 Novel cryptic donor splice site 5-bp upstream, PTC in exon12 (C) PTC in exon 12 Out-of-frame exon 17 skipping, PTC in exon 18 (C)
Underlined mutations are new variants in this study. a Putative protein domain prediction is based on the position of the equivalent residue within the structure of SERCA1. b Splice site mutation in mRNA protein level is demonstrated by RT-PCR analysis (R) or a computational tool (C).
Six new ATP2C1 mutations in Hailey—Hailey disease upstream, both of which lead to PTCs and therefore predict absence or marked reduction of mutated ATP2C1 via nonsense-mediated mRNA decay. Analysis using this tool also revealed that the c.360 + 1G > C mutation resulted in a loss of a donor splice site, thus predicting in-frame skipping of exon 5. In fact, RTPCR analysis of the c.360 + 1G > C mutation in patient 1 revealed a mutant transcript with in-frame skipping of exon 5, as well as expression of the normal transcript. Exon 5 is included in the predicted M2 helix of the molecule, whose functional involvement is described above. The lack of M2 helix may play an important role for the clinical appearance of patient 2, whereas mutated hSPCA1 protein may retain the structure and function of the three cytoplasmic domains. These findings demonstrate the expression of the in-frame shorter transcript without exon 5 through the c.360 + 1G > C mutation, which may account for the mild phenotype. This observation also further supports the theory that haploinsufficiency of ATP2C1 is a prevalent mechanism for the dominant inheritance of HHD. No ATP2C1 mutation was detected in patient 8 with typical HHD. As there is no evidence of a large gene deletion, the disease could be caused by a variant occurring within 50 or 30 untranslated, or intronic, regions not screened in this study, or, alternatively, by a small deletion in the gene, causing the absence of PCR amplification in one allele. It is also possible that this might be another unknown gene mutation related to this patient, although no evidence of genetic heterogeneity has been found in HHD thus far. The hSPCA1 actively pumps Ca2+ from the cytosol to the Golgi apparatus, thus contributing to the maintenance of a low cytosolic Ca2+ concentration in resting conditions [20]. Elevated cytoplasmic Ca2+ caused by the hSPCA1 mutants might act by altering post-translation modifications such as glycosylation, folding, trafficking and/or sorting of key molecules involved in cell-to-cell adhesion. This may cause an inability to maintain structurally intact desmosomes, leading to the acantholysis characteristic of HHD [30]. In this study, we have elucidated the molecular basis of HHD in seven unrelated Japanese and Korean patients. The results further expand the mutation spectrum in HHD and demonstrate the importance of RNA analysis for understanding genotype—phenotype correlations more precisely.
Acknowledgments We thank the patients for their participation. This work was supported by a Grant-in-Aid for Scientific
35 Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, by Health Science Grants for Research on Scientific Disease from the Ministry of Health, Labor and Welfare of Japan, and by an Open Research Center Project of the Ministry of Education, Culture, Sports, Science and Technology of Japan.
References [1] Sakuntabhai A, Ruiz-Perez V, Carter S, et al. Mutations in ATP2A2, encoding a Ca2+ pump, cause Darier disease. Nat Genet 1999;21:271—7. [2] Hu Z, Bonifas JM, Beech J, Bench G, Shigihara T, Ogawa H, Ikeda S, Mauro T, Epstein Jr EH. Mutations in ATP2C1, encoding a calcium pump, cause Hailey—Hailey disease. Nat Genet 2000;24:61—5. [3] Sudbrak R, Brown J, Dobson-Stone C, et al. Hailey—Hailey disease is caused by mutations in ATP2C1 encoding a novel Ca2+ pump. Hum Mol Genet 2000;9:1131—40. [4] Foggia L, Hovnanian A. Calcium pump disorders of the skin. Am J Med Genet C Semin Med Genet 2004;131:C20—31. [5] Ikeda S, Shigihara T, Mayuzumi N, Yu X, Ogawa H. Mutations of ATP2C1 in Japanese patients with Hailey—Hailey disease: intrafamilial and interfamilial phenotype variations and lack of correlation with mutation patterns. J Invest Dermatol 2001;117:1654—6. [6] Chao SC, Tsai YM, Yang MH. Mutation analysis of ATP2C1 gene in Taiwanese patients with Hailey—Hailey disease. Br J Dermatol 2002;146:595—600. [7] Dobson-Stone C, Fairclough R, Dunne E, et al. Hailey—Hailey disease: molecular and clinical characterization of novel mutations in the ATP2C1 gene. J Invest Dermatol 2002;118:338—43. [8] Yokota K, Yasukawa K, Shimizu H. Analysis of ATP2C1 gene mutation in 10 unrelated Japanese families with Hailey— Hailey disease. J Invest Dermatol 2002;118:550—1. [9] Li H, Sun XK, Zhu XJ. Four novel mutations in ATP2C1 found in Chinese patients with Hailey—Hailey disease. Br J Dermatol 2003;149:471—4. [10] Fairclough RJ, Dode L, Vanoevelen J, Andersen JP, Missiaen L, Raeymaekers L, Wuytack F, Hovnanian A. Effect of Hailey—Hailey disease mutations on the function of a new variant of human secretory pathway Ca2+/Mn2+-ATPase (hSPCA1). J Biol Chem 2003;278:24721—30. [11] Fairclough RJ, Lonie L, Van Baelen K, Haftek M, Munro CS, Burge SM, Hovnanian A. Hailey—Hailey disease: identification of novel mutations in ATP2C1 and effect of missense mutation A528P on protein expression levels. J Invest Dermatol 2004;123:67—71. [12] Majore S, Biolcati G, Barboni L, Cannistraci C, Binni F, Crisi A, Picardo M, Grammatico P. ATP2C1 gene mutation analysis in Italian patients with Hailey—Hailey disease. J Invest Dermatol 2005;125:933—5. [13] Ra ´cz E, Csiko ´s M, Ka ´rpa ´ti S. Novel mutations in the ATP2C1 gene in two patients with Hailey—Hailey disease. Clin Exp Dermatol 2005;30:575—7. [14] Ohtsuka T, Okita H, Hama N, Yamazaki S. Novel mutation in ATP2C1 gene in a Japanese patient with Hailey—Hailey disease. Dermatology 2006;212:194—7. [15] Yokota K, Sawamura D. Hailey—Hailey disease with affective disorder: report of a case with novel ATP2C1 gene mutation. J Dermatol Sci 2006;43:150—1.
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[16] Zhang XQ, Wu HZ, Li BX, et al. Mutations in the ATP2C1 gene in Chinese patients with Hailey—Hailey disease. Clin Exp Dermatol 2006;31:702—5. [17] Li X, Xiao S, Peng Z, Liu Y, Wang J, Zhou X. Two novel mutations of the ATP2C1 gene in Chinese patients with Hailey—Hailey disease. Arch Dermatol Res 2007;299:209—11. [18] Zhu YG, Yang S, Gao M, et al. Two novel mutations of the ATP2C1 gene in Chinese families with Hailey—Hailey disease. J Dermatol Sci 2006;42:125—7. [19] Zhang F, Yan X, Jiang D, Tian H, Wang C, Yu L. Eight novel mutations of ATP2C1 identified in 17 Chinese families with Hailey—Hailey disease. Dermatology 2007;215:277—83. [20] Behne MJ, Tu CL, Aronchik I, Epstein E, Bench G, Bikle DD, Pozzan T, Mauro TM. Human keratinocyte ATP2C1 localizes to the Golgi and controls Golgi Ca2+ stores. J Invest Dermatol 2003;121:688—94. [21] Sheffield VC, Cox DR, Lerman LS, Myers RM. Attachment of a 40-base-pair G + C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes. Proc Natl Acad Sci USA 1989;86:232—6. [22] Toyoshima C, Nakasako M, Nomura H, Ogawa H. Crystal structure of the calcium pump of sarcoplasmic reticulum ˚ resolution. Nature 2000;405:647—55. at 2.6 A
[23] Toyoshima C, Nomura H. Structural changes in the calcium pump accompanying the dissociation of calcium. Nature 2002;418:605—11. [24] Olesen C, Sørensen TL, Nielsen RC, Møller JV, Nissen P. Dephosphorylation of the calcium pump coupled to counterion occlusion. Science 2004;306:2251—5. [25] Sorensen TL, Moller JV, Nissen P. Phosphoryl transfer and calcium ion occlusion in the calcium pump. Science 2004;304:1672—5. [26] Toyoshima C, Mizutani T. Crystal structure of the calcium pump with a bound ATP analogue. Nature 2004;430:529— 35. [27] Toyoshima C, Nomura H, Tsuda T. Lumenal gating mechanism revealed in calcium pump crystal structures with phosphate analogues. Nature 2004;432:361—8. [28] Jensen AM, Sørensen TL, Olesen C, Møller JV, Nissen P. Modulatory and catalytic modes of ATP binding by the calcium pump. EMBO J 2006;25:2305—14. [29] Wessagowit V, Nalla VK, Rogan PK, McGrath JA. Normal and abnormal mechanisms of gene splicing and relevance to inherited skin diseases. J Dermatol Sci 2005;40:73—84. [30] Missiaen L, Raeymaekers L, Dode L, et al. SPCA1 pumps and Hailey—Hailey disease. Biochem Biophys Res Commun 2004;322:1204—13.
Available online at www.sciencedirect.com
www.intl.elsevierhealth.com/journals/jods
Molecular and clinical characterization in Japanese and Korean patients with Hailey—Hailey disease: Six new mutations in the ATP2C1 gene Takahiro Hamada a,*, Shunpei Fukuda a, Sachiko Sakaguchi a, Shinichiro Yasumoto a, Soo-Chan Kim b, Takashi Hashimoto a a
Department of Dermatology, Kurume University School of Medicine, Kurume, Japan Department of Dermatology and Cutaneous Biology Research Institute, Yonsei University College of Medicine, Yongdong Severance Hospital, Seoul, Republic of Korea
b
Received 10 January 2008; received in revised form 4 February 2008; accepted 11 February 2008
KEYWORDS Familial benign chronic pemphigus; Acantholysis; P-type ATPase; Ca2+; M2 helix
Summary Background: The autosomal dominant disorder Hailey—Hailey disease (HHD) results from mutations in the ATP2C1 gene, which encodes the human secretory pathway Ca2+/Mn2+-ATPase protein 1. To date, over 90 pathological mutations scattered throughout ATP2C1 have been described with no indication of mutational hotspots or clustering of mutations. No paradigm for genotype—phenotype correlation has emerged. Objectives: To determine the pathogenic ATP2C1 abnormality in additional patients with HHD in order to provide further contributions to the understanding of the molecular basis of this disorder and to add the data to the known mutation database. Methods: In this study, we investigated eight unrelated Japanese and Korean patients with HHD. We performed direct nucleotide sequencing of the ATP2C1 gene in all patients and RT-PCR analysis, using RNA extracted from a skin biopsy, in a patient with the mildest clinical features. Results: We identified seven different heterozygous mutations in seven of the eight investigated patients, including three new single nucleotide deletion/duplication mutations: c.520delC; c.681dupA; c.956delC, three new donor splice site mutations: c.360 + 1G > C; c.899 + 1G > T; c.1570 + 2T > C, as well as a previously described nonsense mutation: p.Arg153X. RT-PCR analysis in the mildest affected patient with a heterozygous c.360 + 1G > C mutation, demonstrated expression of a short in-frame mutant transcript with exon 5 skipping, which may account for the mild phenotype.
* Corresponding author. Tel.: +81 942 31 7571; fax: +81 942 34 2620. E-mail address: [email protected] (T. Hamada). 0923-1811/$30.00 # 2008 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2008.02.003
32
T. Hamada et al. Conclusions: The results expand the known mutation spectrum in HHD and show the importance of RNA analysis for understanding the genotype—phenotype correlations more precisely. # 2008 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction Hailey—Hailey disease (HHD; MIM 169600) is a rare autosomal dominant disorder characterized by abnormal keratinocyte adhesion in the suprabasal layers of the epidermis (acantholysis). The disorder, which usually presents in the third or fourth decade, demonstrates recurrent vesicular lesions, crusted erosions, and warty papules mainly on the neck, axillae, groin, and perineum. Lesions are induced or exacerbated by external factors such as sweating, frictions, and cutaneous infections. Ultrastructural studies of acantholytic cells in HHD reveal perinuclear aggregates of keratin intermediate filaments that have retracted from desmosomal plaques. Darier disease (DD; MIM 124200) is a similarly inherited skin disorder, which has clinical and histological overlap with HHD. DD was shown to be caused by mutations in the ATP2A2 gene on 12q24.1, which encodes the sacro/endoplasmic reticulum Ca2+ ATPase isoform 2 (SERCA2) [1]. Shortly after that report appeared, the molecular basis of HHD was shown to result from mutations in the ATP2C1 gene on 3q22.1, encoding the human secretory pathway Ca2+/Mn2+-ATPase protein 1 (hSPCA1) [2,3]. These proteins belong to the P-type ATPase superfamily, which is defined by the highly conserved phosphorylation sequence DKTGT [4]. No evidence of genetic heterogeneity in HHD has been found thus far. Over 90 pathological mutations scattered throughout the ATP2C1 gene have been described with no indication of mutational hotspots or clustering of mutations [2,3,5—19]. The hSPCA1 is located in the Golgi apparatus in human keratinocytes and serves to actively pump Ca2+ out of the cytoplasm [20]. These findings suggest that intracellular Ca2+ stores play an important role in regulation of epidermal cell—cell adhesion and differentiation. In this study, we investigated the ATP2C1 gene pathology in eight additional unrelated Japanese and Korean patients with HHD in order to provide further contributions to the understanding of the molecular basis of this disorder. We identified seven different mutations in seven of eight investigated familial or sporadic cases. Six of them were new mutations. Furthermore, we performed reverse transcription-polymerase chain reaction (RT-PCR) analysis in a patient with the mildest clinical fea-
tures to assess genotype—phenotype correlation in an ATP2C1 mutation resulting in aberrant splicing.
2. Materials and methods 2.1. PCR amplification of genomic DNA and mutation detection All described studies were performed following the guidelines of the medical ethical committee of Kurume University School of Medicine. Written informed consent was obtained from each individual, and the study was conducted according to the Declaration of Helsinki Principles. Genomic DNA from all individuals was extracted from peripheral blood samples using standard methods. For mutation analysis, PCR fragments were amplified with 28 pairs of primers designed for all exons of the ATP2C1 gene with upstream and downstream primers extending at least 50 bp into flanking introns. The mutation detection strategy consisted of heteroduplex scanning by denaturing gradient gel electrophoresis [21]. The corresponding PCR products showing heteroduplexes were then sequenced directly using Big Dye labeling in an ABI310 genetic analyser (Applied Biosystems, Foster City, USA). Potential mutations were confirmed by restriction endonuclease digestion or bi-directional sequencing and assessed by examining 100 control DNA samples, selected to match the patients’ ethnic backgrounds.
2.2. Genotyping Genotyping was performed by PCR amplification of genomic DNA using the fluorescent microsatellite markers D3S3514, D3S1596, D3S1587, and D3S1292, as described previously [3]. PCR products was then analysed on an ABI310 genetic analyser using Genescan 3.1.2 and Genotyper 2.5.2 software (Applied Biosystems).
2.3. RNA extraction and RT-PCR analysis RNA was only available from patient 1 in this study. Total RNA was isolated from a lesional groin skin sample using a commercial extraction kit (RNeasy Mini Kit, Qiagen K.K., Tokyo, Japan). RT was performed using another commercial kit (SuperScriptTM
Six new ATP2C1 mutations in Hailey—Hailey disease
33
Table 1 Clinical characterization in patients with HHD in this study Patients
Nationality
Sex
Age
Inheritance
Age at onset a
Affected skin lesion(s)
Severity
1 2 3 4 5 6 7
Japanese Japanese Japanese Korean Japanese Japanese Korean
M M M F M M F
57 42 47 62 44 58 55
Sporadic Sporadic Sporadic Familial Familial Sporadic Sporadic
50 20 30 40 20 40 20
Mild Severe Severe Moderate Moderate Moderate Severe
8
Japanese
F
52
Sporadic
40
Groin Axillae, back, and groin Axillae, groin, and perineum Axillae and groin Axillae, groin, and popliteus Axillae, groin, and perineum Neck, axillae, chest, groin, and cubital area Neck, axillae, cubital area, groin, and popliteus
a
Severe
Age at onset, 20, 10—20 years; 30, 30—40 years; 40, 40—50 years; 50, 50—60 years of age.
III First-Strand Synthesis System for RT-PCR, Invitrogen, Carlsbad, CA, USA). A pair of primers for RT-PCR extending across the site of the heterozygous c.360 + 1G > C mutation comprised: forward primer 50 -GCATCAGTTTGATGATGCCG-30 (GenBank NM_014382) and reverse primer 50 -GCGTAAGTCAGCAGGAACTC-30 . The RT-PCR products were subcloned into a PCR-compatible cloning vector (TOPO TA Cloning Kit, Invitrogen) and sequenced directly using Big Dye labeling in an ABI 310 genetic analyser (Applied Biosystems).
Table 2. Patients 3, 4, and 6 were heterozygous for single nucleotide deletion/duplication mutations (c.520delC, c.681dupA, c.956delC), which lead to frameshift and downstream premature termination codons (PTCs). Patients 1, 5, and 7 were heterozygous for donor splice site mutations (c.360 + 1G > C, c.899 + 1G > T, c.1570 + 2T > C) (Fig. 1b). Patient 2 was heterozygous for a nonsense mutation (p.Arg153X). Six of these mutations have not been reported previously. None of the mutations were found in 100 ethnically matched control DNA samples.
3. Results
3.3. RT-PCR analysis in the mild phenotype case reveals an aberrant mutant transcript with in-frame exon 5 skipping
3.1. Clinical features Two familial and six sporadic HHD cases were included in this study. In each patient, diagnosis was established by dermatologists on the basis of clinical and histopathological features. In the two familial cases, we only examined each proband. The patient profiles are summarized in Table 1. Patients 4 and 7 were of Korean origin, and the others were of Japanese origin. The ages at onset were distributed between 20 and 50 years. All patients except patient 1 demonstrated typical clinical features including scaly erythematous plaques, vesiculopustules, and painful erosions on the neck, axillae, chest, cubital area, groin, perineum, and popliteus. Patient 1 showed dusky erythema and papules with tiny erosions restricted to the groin (Fig. 1a). There were no skin abnormalities in other intertrigenous areas.
The mildest clinical manifestations were noted in patient 1, who was heterozygous for the mutation c.360 + 1G > C in intron 5. In cDNA from skin of patient 1, RT-PCR across the site of the c.360 + 1G > C mutation identified two bands of 232 and 196 bp compared with a single 232-bp band in the normal control sample (Fig. 1c). Subcloning and direct sequencing disclosed that the 196-bp band was an aberrant mutant transcript with inframe exon 5 skipping (Fig. 1c). The 232-bp band was the normal wild type transcript. These findings demonstrate the expression of the short in-frame mutant transcript with exon 5 skipping through the c.360 + 1G > C mutation, which may account for the mild phenotype.
3.2. Delineation of pathogenic ATP2C1 mutations in HHD
4. Discussion
Sequencing of genomic DNA in eight unrelated patients with HHD disclosed pathogenic mutations in each patient except patient 8, as detailed in
Among the P-type ATPase superfamily members, SERCA1 has been structurally and functionally well presented. The SERCA1 comprises three cytoplasmic
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Fig. 1 Clinical features and molecular basis of patient 1. (a) Dusky erythema and papules with tiny erosions restricted to the groin. (b) Direct sequencing of genomic DNA of ATP2C1 shows a heterozygous G > C transition at the + 1 position of the exon 5 donor splice site, designated c.360 + 1G > C. (c) RT-PCR, subcloning and direct sequencing across the site of the c.360 + 1G > C mutation reveals a mutant transcript with in-frame exon 5 skipping (196 bp), as well as the normal transcript (232 bp).
domains, designated as A (actuator), N (nucleotidebinding), and P (phosphorylation) domains, and ten (M1—10) transmembrane helices [22]. Two high-affinity Ca2+-binding sites are located side-by-side within the transmembrane region, formed by M4— 6 and M8 helices. Both hSPCA1 and SERCA2 are thought to have similar structures and functions as SERCA1. Six crystal structures are now known that represent different states of the reaction cycle of ATPase [22—28]. For example, during the reaction cycle the cytoplasmic domains form a compact headpiece, then the A domain is tilted by 308 around an axis approximately parallel to the membrane, while the ATPase is phosphorylated [27]. Because the A domain is directly linked to M1, M2, and M3 helices, its movement inevitably causes rearrangement of these helices and leads to opening
of the lumenal gate and releasing the bound Ca2+ into the lumen. A number of mutations scattered throughout the ATP2C1 gene have been described. About 70% of them are nonsense, insertion/deletion or splice site mutations [7]. In this study, we have identified six new heterozygous frameshift or splice site mutations in the ATP2C1 gene in patients with HHD, as detailed in Table 2. The utility of computational tools that can accurately predict cryptic splicing, such as Splice Site Prediction by Neural Network (http://www.fruitfly.org/seq_tools/splice.html), has been described recently [29]. An analysis using this tool revealed that the c.1570 + 2T > C mutation resulted in a loss of a donor splice site, predicting out-of-frame exon 17 skipping; and the c.899 + 1G > T mutation resulted in a novel cryptic donor splice site 5-bp
Table 2 ATP2C1 mutations in patients with HHD in this study Patients
Mutation
Location
Putative protein domain a
Mutation in mRNA protein level b
1 2 3 4 5
c.360 + 1G > C p.Arg153X c.520delC c.681dupA c.899 + 1G > T
intron 5 exon 7 exon 7 exon 8 intron 11
M2 Actuator Actuator Actuator M4
6 7
c.956delC c.1570 + 2T > C
exon 12 intron 17
M4 Nucleotide
In-frame exon 5 skipping (R) PTC in exon 7 PTC in exon 8 PTC in exon 9 Novel cryptic donor splice site 5-bp upstream, PTC in exon12 (C) PTC in exon 12 Out-of-frame exon 17 skipping, PTC in exon 18 (C)
Underlined mutations are new variants in this study. a Putative protein domain prediction is based on the position of the equivalent residue within the structure of SERCA1. b Splice site mutation in mRNA protein level is demonstrated by RT-PCR analysis (R) or a computational tool (C).
Six new ATP2C1 mutations in Hailey—Hailey disease upstream, both of which lead to PTCs and therefore predict absence or marked reduction of mutated ATP2C1 via nonsense-mediated mRNA decay. Analysis using this tool also revealed that the c.360 + 1G > C mutation resulted in a loss of a donor splice site, thus predicting in-frame skipping of exon 5. In fact, RTPCR analysis of the c.360 + 1G > C mutation in patient 1 revealed a mutant transcript with in-frame skipping of exon 5, as well as expression of the normal transcript. Exon 5 is included in the predicted M2 helix of the molecule, whose functional involvement is described above. The lack of M2 helix may play an important role for the clinical appearance of patient 2, whereas mutated hSPCA1 protein may retain the structure and function of the three cytoplasmic domains. These findings demonstrate the expression of the in-frame shorter transcript without exon 5 through the c.360 + 1G > C mutation, which may account for the mild phenotype. This observation also further supports the theory that haploinsufficiency of ATP2C1 is a prevalent mechanism for the dominant inheritance of HHD. No ATP2C1 mutation was detected in patient 8 with typical HHD. As there is no evidence of a large gene deletion, the disease could be caused by a variant occurring within 50 or 30 untranslated, or intronic, regions not screened in this study, or, alternatively, by a small deletion in the gene, causing the absence of PCR amplification in one allele. It is also possible that this might be another unknown gene mutation related to this patient, although no evidence of genetic heterogeneity has been found in HHD thus far. The hSPCA1 actively pumps Ca2+ from the cytosol to the Golgi apparatus, thus contributing to the maintenance of a low cytosolic Ca2+ concentration in resting conditions [20]. Elevated cytoplasmic Ca2+ caused by the hSPCA1 mutants might act by altering post-translation modifications such as glycosylation, folding, trafficking and/or sorting of key molecules involved in cell-to-cell adhesion. This may cause an inability to maintain structurally intact desmosomes, leading to the acantholysis characteristic of HHD [30]. In this study, we have elucidated the molecular basis of HHD in seven unrelated Japanese and Korean patients. The results further expand the mutation spectrum in HHD and demonstrate the importance of RNA analysis for understanding genotype—phenotype correlations more precisely.
Acknowledgments We thank the patients for their participation. This work was supported by a Grant-in-Aid for Scientific
35 Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, by Health Science Grants for Research on Scientific Disease from the Ministry of Health, Labor and Welfare of Japan, and by an Open Research Center Project of the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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