Brain-specific heterozygous loss-of-function of ATP2A2, endoplasmic reticulum Ca2+ pump responsible for Darier's disease, causes behavioral abnormalities and a hyper-dopaminergic state.

A report of a family of Darier's disease with mood disorders drew attention when the causative gene was identified as ATP2A2 (or SERCA2), which encodes a Ca2+ pump on the endoplasmic reticulum (ER) membrane and is important for intracellular Ca2+ signaling. Recently, it was found that loss-of-function mutations of ATP2A2 confer a risk of neuropsychiatric disorders including depression, bipolar disorder and schizophrenia. In addition, a genome-wide association study found an association between ATP2A2 and schizophrenia. However, the mechanism of how ATP2A2 contributes to vulnerability to these mental disorders is unknown. Here, we analyzed Atp2a2 heterozygous brain-specific conditional knockout (hetero cKO) mice. The ER membranes prepared from the hetero cKO mouse brain showed decreased Ca2+ uptake activity. In Atp2a2 heterozygous neurons, decays of cytosolic Ca2+ level were slower than control neurons after depolarization. The hetero cKO mice showed altered behavioral responses to novel environments and impairments in fear memory, suggestive of enhanced dopamine signaling. In vivo dialysis demonstrated that extracellular dopamine levels in the NAc were indeed higher in the hetero cKO mice. These results altogether indicate that the haploinsufficiency of Atp2a2 in the brain causes prolonged cytosolic Ca2+ transients, which possibly results in enhanced dopamine signaling, a common feature of mood disorders and schizophrenia. These findings elucidate how ATP2A2 mutations causing a dermatological disease may exert their pleiotropic effects on the brain and confer a risk for mental disorders.

Protein aggregation of SERCA2 mutants associated with Darier disease elicits ER stress and apoptosis in keratinocytes.

Mutations in sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) underlie Darier disease (DD), a dominantly inherited skin disorder characterized by loss of keratinocyte adhesion (acantholysis) and abnormal keratinization (dyskeratosis) resulting in characteristic mucocutaneous abnormalities. However, the molecular pathogenic mechanism by which these changes influence keratinocyte adhesion and viability remains unknown. We show here that SERCA2 protein is extremely sensitive to endoplasmic reticulum (ER) stress, which typically results in aggregation and insolubility of the protein. Depletion of ER calcium stores is not necessary for the aggregation but accelerates the progression. Systematic analysis of diverse mutants identical to those found in DD patients demonstrated that the ER stress initiator is the SERCA2 mutant protein itself. These SERCA2 proteins were found to be less soluble, to aggregate and to be more polyubiquitinylated. After transduction into primary human epidermal keratinocytes, mutant SERCA2 aggregates elicited ER stress, caused increased numbers of cells to round up and detach from the culture plate, and induced apoptosis. These mutant induced events were exaggerated by increased ER stress. Furthermore, knockdown SERCA2 in keratinocytes rendered the cells resistant to apoptosis induction. These features of SERCA2 and its mutants establish a mechanistic base to further elucidate the molecular pathogenesis underlying acantholysis and dyskeratosis in DD.

Darier disease : a disease model of impaired calcium homeostasis in the skin.

The importance of extracellular calcium in epidermal differentiation and intra-epidermal cohesion has been recognized for many years. Darier disease (DD) was the first genetic skin disease caused by abnormal epidermal calcium homeostasis to be identified. DD is characterized by loss of cell-to-cell adhesion and abnormal keratinization. DD is caused by genetic defects in ATP2A2 encoding the sarco/endoplasmic reticulum Ca(2+)-ATPase isoform 2 (SERCA2). SERCA2 is a calcium pump of the endoplasmic reticulum (ER) transporting Ca(2+) from the cytosol to the lumen of ER. ATP2A2 mutations lead to loss of Ca(2+) transport by SERCA2 resulting in decreased ER Ca(2+) concentration in Darier keratinocytes. Here, we review the role of SERCA2 pumps and calcium in normal epidermis, and we discuss the consequences of ATP2A2 mutations on Ca(2+) signaling in DD. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Darier disease in Slovenia: spectrum of ATP2A2 mutations and relation to patients' phenotypes.

ATP2A2 encodes the sarco/endoplasmic reticulum Ca2+- ATPase (SERCA2) and has been identified as a defective gene in Darier disease (DD). It is an autosomal dominant genodermatosis, which is characterized by loss of adhesion between suprabasal epidermal keratinocytes (acantholysis) and abnormal keratinization (dyskeratosis). We examined 28 Slovenian patients with DD (the cohort of patients represents over 50% of all DD patients in Slovenia) and screened genomic DNA for ATP2A2 mutations and RNA for splice site mutations. We identified 7 different ATP2A2 mutations, 4 of which are novel: A516P, R559G, 544+1del6, and 1762-6del18. We also found two previously described polymorphisms 2741+54 G>A in intron XVIII and 2172 G>A (A724A) in exon 15, with allele frequencies of 64.2% and 11.3%, respectively. The mutations are scattered throughout the gene and affect the actuator, phosphorylation, stalk and transmembrane domains of SERCA2. A P160L mutation in a Slovene patient with severe DD and a history of deafness is another consistent genotype-phenotype correlation. It seems that mutations of the ATP2A2 gene may also play a role in the pathogenesis of deafness, which seems to be a new phenotypic characteristic of DD patients.

Darier disease is associated with heart failure: a cross-sectional case-control and population based study.

Human data supporting a role for endoplasmic reticulum (ER) stress and calcium dyshomeostasis in heart disease is scarce. Darier disease (DD) is a hereditary skin disease caused by mutations in the ATP2A2 gene encoding the sarcoendoplasmic-reticulum Ca2+ ATPase isoform 2 (SERCA2), which causes calcium dyshomeostasis and ER stress. We hypothesized that DD patients would have an increased risk for common heart disease. We performed a cross-sectional case-control clinical study on 25 DD patients and 25 matched controls; and a population-based cohort study on 935 subjects with DD and matched comparison subjects. Main outcomes and measures were N-terminal pro-brain natriuretic peptide, ECG and heart diagnosis (myocardial infarction, heart failure and arrythmia). DD subjects showed normal clinical heart phenotype including heart failure markers and ECG. The risk for heart failure was 1.59 (1,16-2,19) times elevated in DD subjects, while no major differences were found in myocardial infarcation or arrhythmias. Risk for heart failure when corrected for cardivascular risk factors or alcohol misuse was 1.53 (1.11-2.11) and 1.58 (1,15-2,18) respectively. Notably, heart failure occurred several years earlier in DD patients as compared to controls. We conclude that DD patients show a disease specific increased risk of heart failure which should be taken into account in patient management. The observation also strenghtens the clinical evidence on the important role of SERCA2 in heart failure pathophysiology.

Five mutations of ATP2A2 gene in Chinese patients with Darier's disease and a literature review of 86 cases reported in China.

Darier's disease (DD) is an autosomal dominantly inherited skin disorder characterized by loss of adhesion between epidermal cells (acantholysis) and abnormal keratinization. To date, at least 140 mutations in the ATP2A2 gene have been identified as the genetic basis of DD. Here we reported three familial and two sporadic Chinese DD patients totally with four missense mutations (N767D, M494I, M494L, C318F) and one splice-site mutation (1288-6A-->G) in ATP2A2 gene, and presented a literature review of DD cases reported in China since 1989. Our data add new variants to the repertoire of ATP2A2 gene in DD and confirms that most mutations in the ATP2A2 gene are private and missense type. Likewise, the literature review indicates that DD is not uncommon in China and presents more information about genotype-phenotype correlations.

The effect of 13-cis retinoic acid on epidermal lysosomal hydrolase activity in Darier's disease and pityriasis rubra pilaris.

Five patients with Darier's disease and 6 patients with pityriasis rubra pilaris were treated with 13-cis-retinoic acid. Extracts of separated epidermis were assayed for extractable protein, lactic dehydrogenase, Cathepsin D, beta glucuronidase and neutral proteinase before beginning therapy and 2, 4 and 8 weeks after therapy had begun. The epidermal extracts from patients with pityriasis rubra pilaris before beginning therapy were similar to extracts from normal control patients. During the course of therapy with 13-cis-retinoic acid, protein extractability, lactic dehydrogenase and neutral proteinase did not change; there was a highly significant decrease in the specific activity of the lysosomal hydrolases Cathepsin D and beta glucuronidase. A similar but less dramatic fall was noted in the Darier's patients taking 13-cis-retinoic acid. Darier's patients also had a decrease in neutral proteinase activity before beginning therapy; the specific activity of this enzyme increased during the course of therapy. 13-cis-retinoic acid does not induce clinical remission by increasing the intracellular concentration of lysosomal enzymes in epidermis in vivo.

Association study of bipolar disorder at the phospholipase A2 gene (PLA2A) in the Darier's disease (DAR) region of chromosome 12q23-q24.1.

We have previously described a pedigree in which affective disorder and Darier's disease cosegregate and, in an independent sample of 45 bipolar pedigrees, reported evidence in support of linkage between a putative susceptibility locus for bipolar disorder and markers in the Darier's disease region of chromosome 12q23-q24.1. The maximum evidence for linkage was given by a polymorphism at the gene encoding secretory phospholipase A2 (PLA2A), a candidate gene for affective disorder. Dawson et al. (Psychiatric Genetics, 5, 177-180) recently reported allelic association with the same polymorphism at PLA2A in a sample of 54 bipolar probands and 56 controls. We have been unable to replicate the association in 127 Caucasian British DSMIII-R bipolar probands and 223 Caucasian British controls. However, on the basis of several lines of evidence discussed in the paper, we believe that this genomic region in general, and the PLA2A gene in particular, are worthy of further study as candidates in the search for susceptibility genes for the functional psychoses.

Novel mutations of the ATP2A2 gene in two families with Darier's disease.

Darier's disease (DD) is an autosomal dominant genodermatology. Mutations in the ATP2A2 gene encoding sarco-endoplasmic reticulum calcium pumping ATPase type 2 (SERCA2) have been identified as the molecular basis of DD. The aim of this study was to report two Chinese pedigree of DD and to explore the genetic mutations. Polymerase chain reaction was carried out to amplify the exons and flanking intron boundaries of the ATP2A2 gene followed by direct sequencing. Two novel missense mutations were identified, a change of C203 to A (A68E) in exon 3 was found in one family and a change of C2759 to T (S920F) in exon 19 in the other, which were located within the transmembrane domain of SERCA2, highly conserved during evolution. The A68E and S920F mutations might be regarded as the causes of the disease in two Chinese families, but these were not tested functionally. Additional functional experiments are necessary to verify the relevance and suitability of these findings for future use in genetic counseling and prenatal diagnosis.

Spectrum of novel ATP2A2 mutations in patients with Darier's disease.

Darier's disease (DD) is an autosomal dominantly inherited skin disorder characterized by loss of adhesion between epidermal cells (acantholysis) and abnormal keratinization. Recently, we identified ATP2A2 encoding the sarco/endoplasmic reticulum Ca(2+)ATPase isoform 2 as the defective gene in DD. Now we report a spectrum of ATP2A2 mutations in 19 families and six sporadic cases with DD and investigate genotype-phenotype correlations. All 21 exons and flanking intron boundaries were amplified and screened for mutations by conformation-sensitive gel electrophoresis and direct sequencing. We identified 24 novel mutations that are scattered throughout the ATP2A2 gene. Two families shared an identical mutation on a common disease-associated haplotype, suggesting inheritance from a common ancestor. The majority of the mutations (54%; 13/24) led to a premature termination codon which further supports the proposal that haploin-sufficiency is a common molecular mechanism for DD. Thirty-eight per cent of mutations (9/24) result in non-conservative amino acid substitutions at highly conserved positions. Two mutations predict mutated polypeptides lacking or carrying additional amino acids. Marked inter- and intrafamilial phenotypic variability of the disease was observed. These results illustrate the considerable diversity of ATP2A2 mutations causing DD and suggest that additional factors are important contributors to the clinical phenotype.

Darier's disease: an immunohistochemical study using antibodies to proteases.

The expression of plasminogen activators, plasminogen and fibrinogen was investigated in 10 specimens of normal skin and in the involved skin of seven males with Darier's disease, using immunohistochemical techniques. Changes in the localization of proteases were found in the involved skin of Darier's disease, plasminogen being expressed by suprabasal cells and fibrinogen being detected in the keratotic plug.

Linkage analysis of keratosis follicularis spinulosa decalvans, and regional assignment to human chromosome Xp21.2-p22.2.

Keratosis follicularis spinulosa decalvans (KFSD) is a rare X-chromosomal disorder. It consists of follicular hyperkeratosis of the skin, scarring alopecia of the scalp, absence of the eyebrows, and corneal degeneration. There is photophobia in childhood, but the symptoms tend to diminish after puberty, and prognosis for vision is good. Some heterozygotes do show clinical symptoms. In a large Dutch pedigree we performed DNA analysis in order to localize the KFSD gene. In 54 individuals, including 21 affected males, RFLP analysis was done using DNA probes covering the X chromosome. Two-point linkage analyses with 19 informative DNA markers revealed significant linkage to DNA probes on Xp21.1-p22.3. The highest lod scores of 5.70 and 4.38 were obtained with DXS41 and DXS16 at a recombination fraction of zero and 4 cM, respectively. Multipoint linkage data place KFSD between DXS16 and DXS269. Our data confirm X linkage of KFSD in this family and tentatively map the gene on Xp22.2-p21.2. Combined with clinical investigation, RFLP analysis may become an important tool in carrier detection.

Gene dosage of the spermidine/spermine N(1)-acetyltransferase ( SSAT) gene with putrescine accumulation in a patient with a Xp21.1p22.12 duplication and keratosis follicularis spinulosa decalvans (KFSD).

Keratosis follicularis spinulosa decalvans (KFSD) or Siemens-1 syndrome is a rare X-linked disease of unknown etiology affecting the skin and the eye. Although most affected families are compatible with X-linked inheritance, KFSD appears to be clinically and genetically heterogeneous. So far, the gene has been mapped to Xp22.13p22.2 in two extended KFSD families. Analysis of additional recombination events in the first Dutch pedigree located the gene to an interval covering approximately 1 Mb between markers DXS7163 and DXS7593/DXS7105, whereas haplotype reconstruction in the second German family positioned the gene outside the previously identified region, proximal to marker DXS274. We report here the molecular characterization of an Xp21.1p22.12 duplication present in a patient affected with dosage-sensitive sex reversal (DSS) and KFSD. The duplicated region includes both the DAX1 gene (previously demonstrated to be responsible for DSS) and the KFSD interval, in which the gene encoding spermidine/spermine N(1)-acetyltransferase ( SSAT) is located. This enzyme catalyzes the N(1)-acetylation of spermidine and spermine and, by the successive activity of polyamine oxidase, the spermine can be converted to spermidine and the spermidine to putrescine. Overexpression of the SSAT enzyme in a mouse model results in putrescine accumulation and a phenotype with skin and hair abnormalities reminiscent of human KFSD. Analysis of polyamine metabolism in the cells of the patient indicated that the levels of metabolites such as putrescine, spermidine and spermine were consistent with the overexpression of the SSAT gene as in the murine model. Thus, we propose that overexpression of SSAT and the consequent putrescine accumulation are involved in the KFSD phenotype, at least in our propositus.

Expression of sarco/endo-plasmic reticulum Ca2+-ATPase type 2 isoforms (SERCA2) in normal human skin and mucosa, and Darier's disease skin.

BACKGROUND: The recent report that mutations in ATP2A2, which encodes the Ca2+ transporting sarco/endo-plasmic reticulum pump type 2 isoforms (SERCA2), cause Darier's disease (DD) suggests that SERCA2 plays an important role in epidermal cell adhesion and differentiation. However, no data exist regarding SERCA2 expression in normal human skin, mucosa and DD. OBJECTIVES: We have therefore investigated SERCA2 expression in normal human skin (40 samples), oral and vaginal mucosa (13 samples) and DD lesional skin (six samples). MATERIALS AND METHODS: These investigations were performed with a mouse monoclonal antibody specific for human SERCA2, using a standard ABC immunoperoxidase technique. RESULTS: SERCA2 was expressed in all specimens. SERCA2 expression was pronounced in the subnuclear aspect of basal epidermal keratinocytes, with variable suprabasal expression. SERCA2 expression was also observed in the infundibulum and outer root sheath of hair follicles; germinative and mature cells of sebaceous glands; secretory coil and duct of eccrine glands; apocrine gland cells, and arrector pili muscle. Fibroblasts and blood vessels (endothelium and muscle) expressed SERCA2, whereas nerves did not. SERCA2 expression was observed throughout oral and vaginal mucosa. In DD skin, strong SERCA2 positivity was detected in the basal, suprabasal and acantholytic lesional cells. Perilesional DD skin was comparable to normal skin. CONCLUSIONS: These findings support the hypothesis that SERCA2 is an important player in cutaneous biology, and provide baseline data that will facilitate the design and interpretation of functional studies of cutaneous SERCA2.

Distinct types of abnormality in kinetic properties of three Darier disease-causing sarco(endo)plasmic reticulum Ca2+-ATPase mutants that exhibit normal expression and high Ca2+ transport activity.

The possible functional abnormalities in three different Darier disease-causing Ca(2+)-ATPase (SERCA2b) mutants, Ile(274) --> Val at the lumenal end of M3, Leu(321) --> Phe on the cytoplasmic part of M4, and Met(719) --> Ile in P domain, were explored, because they exhibited nearly normal expression and localization in COS-1 cells and the high ATPase and coupled Ca(2+) transport activities that were essentially identical (L321F) or slightly lower (I274V by approximately 35% and M719I by approximately 30%) as compared with those of the wild type. These mutations happened to be in Japanese patients found previously by us. Kinetic analyses revealed that each of the mutants possesses distinct types of abnormalities; M719I and L321F possess the 2-3-fold reduced affinity for cytoplasmic Ca(2+), whereas I274V possesses the normal high affinity. L321F exhibited also the remarkably reduced sensitivity to the feedback inhibition of the transport cycle by accumulated lumenal Ca(2+), as demonstrated with the effect of Ca(2+) ionophore on ATPase activity and more specifically with the effects of Ca(2+) (up to 50 mm) on the decay of phosphoenzyme intermediates. The results on I274V and M719I suggest that the physiological requirement for Ca(2+) homeostasis in keratinocytes to avoid haploinsufficiency is very strict, probably much more than considered previously. The insensitivity to lumenal Ca(2+) in L321F likely brings the lumenal Ca(2+) to an abnormally elevated level. The three mutants with their distinctively altered kinetic properties will thus likely cause different types of perturbation of intracellular Ca(2+) homeostasis, but nevertheless all types of perturbation result in Darier disease. It might be possible that the observed unique feature of L321F could possibly be associated with the specific symptoms in the pedigree with this mutation, neuropsychiatric disorder, and behavior problems. The results also provided further insight into the global nature of conformational changes of SERCAs for ATP-driven Ca(2+) transport.

Expression of the sarco/endoplasmic reticulum calcium ATPase type 2 and 3 isoforms in normal skin and Darier's disease.

BACKGROUND: Darier's disease (DD) is caused by mutations in ATP2A2, which encodes the sarco/endoplasmic reticulum calcium ATPase type 2 (SERCA2), a member of a family of calcium pumps important in intracellular calcium signalling. SERCA2 has two isoforms. SERCA2a occurs mainly in cardiac and skeletal muscle, whereas SERCA2b occurs ubiquitously and is coexpressed with the related SERCA type 3 (SERCA3) in many tissues. It is not known why mutations in the widely expressed SERCA2 manifest as a focal skin disease. OBJECTIVES: To provide insight into the pathogenesis of DD by examining SERCA isoform expression in normal skin and DD skin. METHODS: Using immunohistochemistry we studied SERCA2a, SERCA2b and SERCA3 expression in nonlesional and lesional skin from seven patients with DD and normal skin from seven control subjects. We quantified SERCA2a and SERCA2b staining intensity by grey scale analysis of fluorescence intensity. RESULTS: In normal and DD epidermis both SERCA2a and SERCA2b staining was seen. SERCA2a staining in epidermis was less intense relative to pilar muscle whereas SERCA2b staining in epidermis was of marginally greater intensity than in pilar muscle. SERCA3 was not expressed in normal or DD epidermis, but was found in eccrine glands and blood vessels. No reduction was detected in SERCA2a or SERCA2b staining intensity in DD nonlesional epidermis compared with control epidermis. In within-patient comparisons, SERCA2a and SERCA2b staining in lesional epidermis was less intense than in nonlesional epidermis. CONCLUSIONS: Both SERCA2a and SERCA2b are present in epidermis, although the latter may predominate. The absence of coexpressed SERCA3 in epidermis may explain the localization of DD. Comparable SERCA2 staining intensity in nonlesional DD and control epidermis, even in patients predicted to be haploinsufficient, suggests partial compensation by upregulation of the normal allele. Unknown additional factors may trigger focal lesions by overcoming this compensation. Reduced staining intensity in lesional tissue may be secondary, or may reflect local downregulation of SERCA2 expression predisposing to development of focal lesions.

Mutational analysis of phospholipase A2A: a positional candidate susceptibility gene for bipolar disorder.

Evidence for the involvement of genetic factors in the pathogenesis of bipolar affective disorder is now well established. However, the mode of inheritance is non-mendelian and this makes the identification of susceptibility loci difficult. A short-cut to localisation of a disease gene for an oligogenic/multifactorial disorder such as bipolar disorder may come from observation of cosegregation with a monogenic trait. We have described a family (pedigree 324) in which there was cosegregation of major affective disorder and Darier's disease, a dominantly inherited skin disorder, and hypothesised that this reflects genetic linkage between genes involved in these disorders. Genetic mapping studies have placed the locus for Darier's disease on chromosome 12q23-q24. We conducted subsequent linkage studies (1995) upon 45 bipolar families (without Darier's disease). These results showed some evidence in favour of linkage with chromosome 12q markers with maximum evidence at a trinucleotide repeat marker within intron 1 of the phospholipase A2A (PLA2A) gene. Evidence for linkage was more significant when analysing the 22 families comprising the Cardiff centre sample, which were expected to be most genetically similar to pedigree 324.

Darier's disease: from dyskeratosis to endoplasmic reticulum calcium ATPase deficiency.

The skin is the body's largest organ and has an essential barrier protective function against physical, chemical, and pathogen aggressions and prevents fluid loss. The outer layer of the skin, known as the epidermis, plays a key role in this protection, through a tightly regulated differentiation programme from basal keratinocytes to the stratum corneum at the skin surface. During this process, keratinocytes from the base of the epidermis undergo major morphological and functional changes during their migration through the spinous and granular layers, to become terminally differentiated corneocytes which will be shed from the skin's surface. The role of extracellular Ca2+ in cell-to-cell adhesion and in epidermal differentiation was known to be important, but the identification of the sarco/endoplasmic reticulum Ca2+ transport ATPase (ATP2A2) as the defective gene in a rare genetic skin disease known as Darier's disease, came as a surprise and shed light on the key role of Ca2+ signaling in the homeostasis of the epidermis.

Immunolocalization of human gelatinase (type IV collagenase, MMP-9) and tissue inhibitor of metalloproteinase 1 in Hailey-Hailey and Darier's diseases.

BACKGROUND: The formation of lacunae and acantholysis as well as dyskeratosis are characteristic features of Hailey-Hailey disease (HHD) and Darier's disease (DD). Matrix metalloproteinases (MMPs) and their inhibitors like tissue inhibitors of metalloproteinases (TIMPs) have been thought to play major roles in the tissue metabolism. OBJECTIVE: The aim of this study was to investigate the role of MMP-9 and TIMP-1 in HHD and DD. METHODS: We examined localizations of these two molecules by immunostaining using specific monoclonal antibodies. RESULTS: MMP-9 was positively stained in dyskeratotic or detaching cells around lacunae in HHD and DD. TIMP-1 showed a positive staining pattern throughout the epidermis. CONCLUSION: MMP-9 might be involved in the pathophysiological process of HHD and DD in the presence of TIMP-1.