Letters to the Editor
394
Exon 12 of the ATP2A2 gene in patients with Darier disease: one novel mutation and one previously described Editor Darier disease (DD; OMIM: 124200), also known as Darier– White disease, keratosis follicularis, is a rare autosomal dominant heritable disease estimated to affect 1/55 000–1/100 000 individuals.1 It is characterized clinically by warty papules and plaques over the seborrhoeic area, such as central trunk, flexures, scalp and forehead, mostly presenting before the third decade.2 The disorder may present with various clinical manifestations including the nails, oral mucosa and neuropsychiatric abnormalities. DD is caused by mutations in the ATP2A2 gene (MIM: 108740), which encodes the enzyme sarco/endoplasmic reticulum Ca2+ ATPase type 2 (SERCA2-ATPase), a calcium pump in the sarcoplasmic reticulum that utilizes the chemical energy from ATP.3 The ATP2A2 mutations have been detected in patients of varying ethnic backgrounds. Here, we describe the identification of two mutations in ATP2A2 gene in two families with DD. Two three-generation families with DD from Chongqing in China were recruited. Family A consisted of two affected and five unaffected individuals, family B consisted of three affected and seven unaffected individuals. The proband of family A, a 51-year-old man, presented with a 39-year history of itchy and hyperkeratotic papules and plaques on the face (Fig. 1a), upper trunk and extremities. These lesions were exacerbated in hot summer or after sun exposure. The index case in pedigree B was a 47-year-old woman who developed similar skin lesions (Fig. 1b) at the age of 15 years. The diagnosis was confirmed by clinical and histopathological findings.
(a)
The study was approved by Ethical Review Committee of Chongqing hospital of traditional Chinese medicine with written informed consent obtained for every subject. Genomic DNA was extracted using standard procedures from EDTA blood samples. All exons of ATP2A2 gene were amplified by touchdown PCR from genomic DNA. The reaction products purified were sequenced in both directions on an automated DNA sequencer (ABI 3730; Applied Biosystems, Foster City, CA, USA). We identified two heterozygous mutations in exon 12 of the ATP2A2 gene in two different families. A heterozygous A to G transition occurred in nucleotide 1540 in the patients of family A, which results in a lysine to glutamic acid substitution at codon 514 (K514E) (Fig. 2a). A heterozygous C to T transition at nucleotide 1484 (The splice site is indicated by a black vertical bar in Fig. 2) results in a serine to leucine substitution at codon 495 (S495L) (Fig. 2c) in the patients of family B. These two mutations were not detected in unaffected individuals in the two families and the 100 unrelated controls, suggesting that they were not common polymorphisms. The former mutation was novel and the latter has been reported previously. The human ATP2A2 gene, mapped to 12q23–24.1, spans a region of 76 kb, consists of 21 exons, and encodes a 4.5-kb transcript consisting of three cytoplasmic domains and transmembrane helices.4 ATP2A2 defects may affect the expression level of SERCA2b, disturb cell–cell adhesion and keratinocyte differentiation and signalling.5 So far, over 232 pathogenic mutations have been detected within ATP2A2 gene in patients with DD,6 which were scattered along the entire gene without any clustering. The two mutations were located within the phosphorylation region. Intriguingly, the other missense mutations K514R (c.1541 A>G) in the same lysine codon were reported in DD patients associated with neuropsychiatric disorders from Anhui Province in China.7 K514E is the second mutation found to affect this residue. At amino acid position 495, the S495L muta-
(b)
Figure 1 Multiple keratotic papules and plaques on the face of the proband in the family A (a), similar skin lesions on the neck of the proband in the family B (b).
JEADV 2015, 29, 391–401
© 2014 European Academy of Dermatology and Venereology
Letters to the Editor
395
Exon 12
Intron 12
(a)
Exon 12
(b)
Intron 12
(c)
Figure 2 Mutation analysis of exon 12 in ATP2A2 gene in the two patients. (a) Heterozygous missense mutation 1540 A > G in the proband of the family A. (c) Heterozygous missense mutation 1484 C > T in the proband of the family B. (b, d) Partial wild-type sequence of exon 20.
(d)
tion has been previously reported by Sakuntabhai et al.8 and Green et al.;6 two other alternative amino acid substitutions S495F,9 S495X10 have been identified. Taken together, these results indicate that this position is a hot spot for mutations in the ATP2A2 gene. This study confirmed that a few ATP2A2 variants underlying DD may not be as family-specific as first thought.6 Mutational analysis of the ATP2A2 gene will provide a better understanding of the genotype–phenotype correlations of this disease.
Funding sources This work was supported by Medical Scientific Research Project of Chongqing Municipal Health Bureau (2011-2-319).
Acknowledgments The authors gratefully acknowledge the patients and their families for their participation in this study. B.-J. Shi,1* M. Xue,1 Y.-J. Zhu,2 S.-P. Wang,1 Y. Du,2 D.-Y. Chen,2 Q.-C. Diao1,* 1
Department of Dermatology, Chongqing hospital of traditional chinese medicine (the First People’s Hospital of Chongqing City), Chongqing,
JEADV 2015, 29, 391–401
China, 2Department of Dermatology, the Affiliated Hospital of Luzhou Medical College, Luzhou, China, *Correspondence: Q.-C. Diao and B.-J. Shi. E-mails: [email protected]; [email protected]
References 1 Cooper SM, Burge SM. Darier’s disease: epidemiology, pathophysiology, and management. Am J Clin Dermatol 2003; 4: 97–105. 2 Burge SM, Wilkinson JD. Darier-White disease: a review of the clinical features in 163 patients. J Am Acad Dermatol 1992; 27: 40–50. 3 Dhitavat J, Dode L, Leslie N et al. Mutations in the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase isoform cause Darier’s disease. J Invest Dermatol 2003; 121: 486–489. 4 Ikeda S, Wakem P, Haake A et al. Localization of the gene for Darier disease to a 5-cM intervalon chromosome 12q. J Invest Dermatol 1994; 103: 478–481. 5 Cho JK, Bikle DD. Decrease of Ca2+ ATPase activity in human keratinocytes during calcium-induced differentiation. J Cell Physiol 1997; 172: 146–154. 6 Green EK, Gordon-Smith K, Burge SM et al. Novel ATP2A2 mutations in a large sample of individuals with Darier disease. J Dermatol 2013; 40: 259–266. 7 Yang S, Sun LD, Liu HS et al. A novel missense mutation ofthe ATP2A2 gene in a Chinese family with Darier’s disease. Arch Dermatol Res 2004; 296: 21–24.
© 2014 European Academy of Dermatology and Venereology
Letters to the Editor
396
8 Sakuntabhai A, Ruiz-Perez V, Carter S et al. Mutations in ATP2A2, encoding a Ca2+ pump, cause Darier disease. Nat Genet 1999; 21: 271–277. 9 Ruiz-Perez VL, Carter SA, Healy E et al. ATP2A2 mutations in Darier’s disease: variant cutaneous phenotypes are associated with missense mutations, but neuropsychiatric features are independent of mutation class. Hum Mol Genet 1999; 8: 1621–1630. 10 Ikeda S, Mayuzumi N, Shigihara T, Epstein EH Jr, Goldsmith LA, Ogawa H. Mutations in ATP2A2 in patients with Darier’s disease. J Invest Dermatol 2003; 121: 475–477. DOI: 10.1111/jdv.12398
Increased levels of matrix metalloproteinase-9 and interleukin-8 in blister fluids of dystrophic and junctional epidermolysis bullosa patients Editor Epidermolysis bullosa (EB) characterizes a group of genodermatoses that affect the integrity of epithelial layers, phenotypically resulting in severe blistering of the skin and mucous membranes. The most severe subtypes are junctional EB (JEB) and dystrophic EB (DEB), which are caused by mutations in different genes encoding structural proteins, which render stability to the skin.
In a recent study, we showed that the gene and protein expression levels of matrix metalloproteinase-9 (MMP-9) and interleukin-8 (CXCL8/IL-8) were increased significantly in cultured keratinocytes of the simplex group of EB (EBS). These data were confirmed in vivo, by analyzing the blister fluids of eight EBS patients1. The role of proteases in the pathophysiology of blistering diseases such as pemphigus vulgaris (PV) has been investigated widely and led to the specific proteolysis hypothesis2. In cell culture experiments and in a mouse model for PV, MMP-9 was shown to be a candidate mediator of acantholysis, the loss of epithelial cell–cell connections3. On the other hand, IL-8, also known as the chemokine CXCL8, was shown to play a major role in the pathogenesis of the autoimmune blistering disease bullous pemphigoid (BP). Further, in a mouse model of BP, the infiltration of neutrophils into the skin was shown to be obligatory for subepidermal blistering, and the infiltration of neutrophils was shown to be dependent on IL-8 activity4. On the basis of the findings obtained in PV, BP and EBS, we hypothesized that MMP-9 and CXCL8/IL-8 play an important role in blister formation in JEB and DEB, as well. We investigated blister fluids of patients suffering from recessive dystrophic epidermolysis bullosa (RDEB, n = 6) and junctional epidermolysis bullosa (JEB, n = 3) (Table 1). The blister fluids of dystrophic and junctional EB-patients contained significantly increased levels of MMP-9 and CXCL8/IL-8 (Fig. 1) with variations between single individuals. Blister fluids of the three
Table 1 Summarized patient data Mutation(s)
Age*
Blister-location
1
Patient MB
Gene
Zygosity
Healthy control
76
Left inguinal
2
MB
Healthy control
42
Left and right heel
3
BB
Healthy control
34
Left foot
4
RDEB-sev gen
COL7A1
Homozygous
6081delC, ex73
13
Left foot
5
RDEB-sev gen
COL7A1
Compound heterozygous
5532 + 1G>A, ex/in 64, as K142R, 425A>G, ex3, as
12
Right knee
6
RDEB-sev gen
COL7A1
Compound heterozygous
682 + 1G>A, ex/in 5, as P1270L, 3809C>T, ex30
22
Pooled**
7
RDEB-sev gen
COL7A1
Compound heterozygous
682 + 1G>A, ex/in 5, as P2385Q, 7154delC, ex93
3
Left thigh
8
RDEB-sev gen
COL7A1
9
RDEB-sev gen
COL7A1
Compound heterozygous
R2332X, 6994C>T, ex90 R2685X, 8053C>T, ex109
10
JEB-nHgen
LAMB3
Compound heterozygous
R635X, 1903C>T, ex14 3009C>T, ex20, as
41
Pooled**
11
JEB-nHgen
COL7A1
Compound heterozygous
Q1403X, 4207C>T, ex53 4003delTC, ex52
22
Pooled**
12
JEB-nHgen
COL7A1
Compound heterozygous
E1163X, 3487G>T, ex49 G1441W, 4319dupC, ex54
4
Forehead
Unknown
19 2
Left knee Left shinbone
*Age (in years) at time of sample collection. **Two or more blisters collected from different locations and pooled. as, altered splicing; BB, burn blister; del, deletion; dup, duplication; ex, exon; in, intron; MB, mechanical blister. Control samples 1, 2 and 3 are identical to those in Ref. [1].
JEADV 2015, 29, 391–401
© 2014 European Academy of Dermatology and Venereology
This document is a scanned copy of a printed document. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material.
394
Exon 12 of the ATP2A2 gene in patients with Darier disease: one novel mutation and one previously described Editor Darier disease (DD; OMIM: 124200), also known as Darier– White disease, keratosis follicularis, is a rare autosomal dominant heritable disease estimated to affect 1/55 000–1/100 000 individuals.1 It is characterized clinically by warty papules and plaques over the seborrhoeic area, such as central trunk, flexures, scalp and forehead, mostly presenting before the third decade.2 The disorder may present with various clinical manifestations including the nails, oral mucosa and neuropsychiatric abnormalities. DD is caused by mutations in the ATP2A2 gene (MIM: 108740), which encodes the enzyme sarco/endoplasmic reticulum Ca2+ ATPase type 2 (SERCA2-ATPase), a calcium pump in the sarcoplasmic reticulum that utilizes the chemical energy from ATP.3 The ATP2A2 mutations have been detected in patients of varying ethnic backgrounds. Here, we describe the identification of two mutations in ATP2A2 gene in two families with DD. Two three-generation families with DD from Chongqing in China were recruited. Family A consisted of two affected and five unaffected individuals, family B consisted of three affected and seven unaffected individuals. The proband of family A, a 51-year-old man, presented with a 39-year history of itchy and hyperkeratotic papules and plaques on the face (Fig. 1a), upper trunk and extremities. These lesions were exacerbated in hot summer or after sun exposure. The index case in pedigree B was a 47-year-old woman who developed similar skin lesions (Fig. 1b) at the age of 15 years. The diagnosis was confirmed by clinical and histopathological findings.
(a)
The study was approved by Ethical Review Committee of Chongqing hospital of traditional Chinese medicine with written informed consent obtained for every subject. Genomic DNA was extracted using standard procedures from EDTA blood samples. All exons of ATP2A2 gene were amplified by touchdown PCR from genomic DNA. The reaction products purified were sequenced in both directions on an automated DNA sequencer (ABI 3730; Applied Biosystems, Foster City, CA, USA). We identified two heterozygous mutations in exon 12 of the ATP2A2 gene in two different families. A heterozygous A to G transition occurred in nucleotide 1540 in the patients of family A, which results in a lysine to glutamic acid substitution at codon 514 (K514E) (Fig. 2a). A heterozygous C to T transition at nucleotide 1484 (The splice site is indicated by a black vertical bar in Fig. 2) results in a serine to leucine substitution at codon 495 (S495L) (Fig. 2c) in the patients of family B. These two mutations were not detected in unaffected individuals in the two families and the 100 unrelated controls, suggesting that they were not common polymorphisms. The former mutation was novel and the latter has been reported previously. The human ATP2A2 gene, mapped to 12q23–24.1, spans a region of 76 kb, consists of 21 exons, and encodes a 4.5-kb transcript consisting of three cytoplasmic domains and transmembrane helices.4 ATP2A2 defects may affect the expression level of SERCA2b, disturb cell–cell adhesion and keratinocyte differentiation and signalling.5 So far, over 232 pathogenic mutations have been detected within ATP2A2 gene in patients with DD,6 which were scattered along the entire gene without any clustering. The two mutations were located within the phosphorylation region. Intriguingly, the other missense mutations K514R (c.1541 A>G) in the same lysine codon were reported in DD patients associated with neuropsychiatric disorders from Anhui Province in China.7 K514E is the second mutation found to affect this residue. At amino acid position 495, the S495L muta-
(b)
Figure 1 Multiple keratotic papules and plaques on the face of the proband in the family A (a), similar skin lesions on the neck of the proband in the family B (b).
JEADV 2015, 29, 391–401
© 2014 European Academy of Dermatology and Venereology
Letters to the Editor
395
Exon 12
Intron 12
(a)
Exon 12
(b)
Intron 12
(c)
Figure 2 Mutation analysis of exon 12 in ATP2A2 gene in the two patients. (a) Heterozygous missense mutation 1540 A > G in the proband of the family A. (c) Heterozygous missense mutation 1484 C > T in the proband of the family B. (b, d) Partial wild-type sequence of exon 20.
(d)
tion has been previously reported by Sakuntabhai et al.8 and Green et al.;6 two other alternative amino acid substitutions S495F,9 S495X10 have been identified. Taken together, these results indicate that this position is a hot spot for mutations in the ATP2A2 gene. This study confirmed that a few ATP2A2 variants underlying DD may not be as family-specific as first thought.6 Mutational analysis of the ATP2A2 gene will provide a better understanding of the genotype–phenotype correlations of this disease.
Funding sources This work was supported by Medical Scientific Research Project of Chongqing Municipal Health Bureau (2011-2-319).
Acknowledgments The authors gratefully acknowledge the patients and their families for their participation in this study. B.-J. Shi,1* M. Xue,1 Y.-J. Zhu,2 S.-P. Wang,1 Y. Du,2 D.-Y. Chen,2 Q.-C. Diao1,* 1
Department of Dermatology, Chongqing hospital of traditional chinese medicine (the First People’s Hospital of Chongqing City), Chongqing,
JEADV 2015, 29, 391–401
China, 2Department of Dermatology, the Affiliated Hospital of Luzhou Medical College, Luzhou, China, *Correspondence: Q.-C. Diao and B.-J. Shi. E-mails: [email protected]; [email protected]
References 1 Cooper SM, Burge SM. Darier’s disease: epidemiology, pathophysiology, and management. Am J Clin Dermatol 2003; 4: 97–105. 2 Burge SM, Wilkinson JD. Darier-White disease: a review of the clinical features in 163 patients. J Am Acad Dermatol 1992; 27: 40–50. 3 Dhitavat J, Dode L, Leslie N et al. Mutations in the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase isoform cause Darier’s disease. J Invest Dermatol 2003; 121: 486–489. 4 Ikeda S, Wakem P, Haake A et al. Localization of the gene for Darier disease to a 5-cM intervalon chromosome 12q. J Invest Dermatol 1994; 103: 478–481. 5 Cho JK, Bikle DD. Decrease of Ca2+ ATPase activity in human keratinocytes during calcium-induced differentiation. J Cell Physiol 1997; 172: 146–154. 6 Green EK, Gordon-Smith K, Burge SM et al. Novel ATP2A2 mutations in a large sample of individuals with Darier disease. J Dermatol 2013; 40: 259–266. 7 Yang S, Sun LD, Liu HS et al. A novel missense mutation ofthe ATP2A2 gene in a Chinese family with Darier’s disease. Arch Dermatol Res 2004; 296: 21–24.
© 2014 European Academy of Dermatology and Venereology
Letters to the Editor
396
8 Sakuntabhai A, Ruiz-Perez V, Carter S et al. Mutations in ATP2A2, encoding a Ca2+ pump, cause Darier disease. Nat Genet 1999; 21: 271–277. 9 Ruiz-Perez VL, Carter SA, Healy E et al. ATP2A2 mutations in Darier’s disease: variant cutaneous phenotypes are associated with missense mutations, but neuropsychiatric features are independent of mutation class. Hum Mol Genet 1999; 8: 1621–1630. 10 Ikeda S, Mayuzumi N, Shigihara T, Epstein EH Jr, Goldsmith LA, Ogawa H. Mutations in ATP2A2 in patients with Darier’s disease. J Invest Dermatol 2003; 121: 475–477. DOI: 10.1111/jdv.12398
Increased levels of matrix metalloproteinase-9 and interleukin-8 in blister fluids of dystrophic and junctional epidermolysis bullosa patients Editor Epidermolysis bullosa (EB) characterizes a group of genodermatoses that affect the integrity of epithelial layers, phenotypically resulting in severe blistering of the skin and mucous membranes. The most severe subtypes are junctional EB (JEB) and dystrophic EB (DEB), which are caused by mutations in different genes encoding structural proteins, which render stability to the skin.
In a recent study, we showed that the gene and protein expression levels of matrix metalloproteinase-9 (MMP-9) and interleukin-8 (CXCL8/IL-8) were increased significantly in cultured keratinocytes of the simplex group of EB (EBS). These data were confirmed in vivo, by analyzing the blister fluids of eight EBS patients1. The role of proteases in the pathophysiology of blistering diseases such as pemphigus vulgaris (PV) has been investigated widely and led to the specific proteolysis hypothesis2. In cell culture experiments and in a mouse model for PV, MMP-9 was shown to be a candidate mediator of acantholysis, the loss of epithelial cell–cell connections3. On the other hand, IL-8, also known as the chemokine CXCL8, was shown to play a major role in the pathogenesis of the autoimmune blistering disease bullous pemphigoid (BP). Further, in a mouse model of BP, the infiltration of neutrophils into the skin was shown to be obligatory for subepidermal blistering, and the infiltration of neutrophils was shown to be dependent on IL-8 activity4. On the basis of the findings obtained in PV, BP and EBS, we hypothesized that MMP-9 and CXCL8/IL-8 play an important role in blister formation in JEB and DEB, as well. We investigated blister fluids of patients suffering from recessive dystrophic epidermolysis bullosa (RDEB, n = 6) and junctional epidermolysis bullosa (JEB, n = 3) (Table 1). The blister fluids of dystrophic and junctional EB-patients contained significantly increased levels of MMP-9 and CXCL8/IL-8 (Fig. 1) with variations between single individuals. Blister fluids of the three
Table 1 Summarized patient data Mutation(s)
Age*
Blister-location
1
Patient MB
Gene
Zygosity
Healthy control
76
Left inguinal
2
MB
Healthy control
42
Left and right heel
3
BB
Healthy control
34
Left foot
4
RDEB-sev gen
COL7A1
Homozygous
6081delC, ex73
13
Left foot
5
RDEB-sev gen
COL7A1
Compound heterozygous
5532 + 1G>A, ex/in 64, as K142R, 425A>G, ex3, as
12
Right knee
6
RDEB-sev gen
COL7A1
Compound heterozygous
682 + 1G>A, ex/in 5, as P1270L, 3809C>T, ex30
22
Pooled**
7
RDEB-sev gen
COL7A1
Compound heterozygous
682 + 1G>A, ex/in 5, as P2385Q, 7154delC, ex93
3
Left thigh
8
RDEB-sev gen
COL7A1
9
RDEB-sev gen
COL7A1
Compound heterozygous
R2332X, 6994C>T, ex90 R2685X, 8053C>T, ex109
10
JEB-nHgen
LAMB3
Compound heterozygous
R635X, 1903C>T, ex14 3009C>T, ex20, as
41
Pooled**
11
JEB-nHgen
COL7A1
Compound heterozygous
Q1403X, 4207C>T, ex53 4003delTC, ex52
22
Pooled**
12
JEB-nHgen
COL7A1
Compound heterozygous
E1163X, 3487G>T, ex49 G1441W, 4319dupC, ex54
4
Forehead
Unknown
19 2
Left knee Left shinbone
*Age (in years) at time of sample collection. **Two or more blisters collected from different locations and pooled. as, altered splicing; BB, burn blister; del, deletion; dup, duplication; ex, exon; in, intron; MB, mechanical blister. Control samples 1, 2 and 3 are identical to those in Ref. [1].
JEADV 2015, 29, 391–401
© 2014 European Academy of Dermatology and Venereology
This document is a scanned copy of a printed document. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material.
