Human Molecular Genetics, 2000, Vol. 9, No. 7 1131-1140
© 2000 Oxford University Press
Hailey–Hailey disease is caused by mutations in ATP2C1 encoding a novel Ca2+ pump
1Max-Planck-Institut für Molekulare Genetik, D-14195 Berlin, Germany, 2The Wellcome Trust Centre for Human Genetics, University of Oxford, OX3 7BN, UK, 3Human Genetics Unit, School of Biochemistry and Genetics, University of Newcastle Newcastle upon Tyne, NE1 7RU, UK, 4Department of Dermatology, Southampton General Hospital, Southampton, SO16 6YD, 5Department of Dermatology, General Hospital, Kandy, Sri Lanka, 6Department of Dermatology, La Miletrie Hospital, Poitiers, France, 7Cabinet Médical Wilson, Limeil-Brévannes, France, 8Department of Dermatology, Southern General Hospital, Glasgow, G51 4TF, UK and 9Department of Dermatology, Churchill Hospital, Oxford, OX3 7LJ, UK
Received 4 January 2000; Revised and Accepted 10 February 2000.
DDBJ/EMBL/GenBank accession numbers: human: ATP2C1a, AF189723; ATP2C1b, AF225981; ATP2A2b, M23114; ATP2B2, M97260; stMPIMG1, AJ250604; stMPIMG4, AJ250607; stMPIMG10, AJ250613; stMPIMG13, AJ250616; stMPIMG15, AJ250618; stMPIMG19, AJ250619; stMPIMG20, AJ250620; rat: SPLA, M93017; yeast: PMR1, M25488..
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSNOTE ADDED IN PROOFREFERENCES |
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Hailey–Hailey disease (HHD) is an autosomal dominant skin disorder characterized by suprabasal cell separation (acantholysis) of the epidermis. Previous genetic linkage studies localized the gene to a 5 cM interval on human chromosome 3q21. After reducing the disease critical region to <1 cM, we used a positional cloning strategy to identify the gene ATP2C1, which is mutated in HHD. ATP2C1 encodes a new class of P-type Ca2+-transport ATPase, which is the homologue for the rat SPLA and the yeast PMR1 medial Golgi Ca2+ pumps and is related to the sarco(endo)plasmic calcium ATPase (SERCA) and plasma membrane calcium ATPase (PCMA) families of Ca2+ pumps. The predicted protein has the same apparent transmembrane organization and contains all of the conserved domains present in other P-type ATPases. ATP2C1 produces two alternative splice variants of ~4.5 kb encoding predicted proteins of 903 and 923 amino acids. We identified 13 different mutations, including nonsense, frameshift insertion and deletions, splice-site mutations, and non-conservative missense mutations. This study dem- onstrates that defects in ATP2C1 cause HHD and together with the recent identification of ATP2A2 as the defective gene in Darier’s disease, provide further evidence of the critical role of Ca2+ signaling in maintaining epidermal integrity.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSNOTE ADDED IN PROOFREFERENCES |
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Hailey–Hailey disease (HHD; OMIM 16960) (familial benign chronic pemphigus) is a blistering dermatosis, which is inherited as an autosomal dominant trait (1–3) and usually presents around the third and fourth decades. Painful erosions, vesico-pustules and scaly erythematous plaques appear at sites of friction such as the sides of the neck, the axillae, the groins and the perineum (Fig. 1a). Painful fissures (rhagades) are common in flexures and may limit mobility. Flexural disease may become hypertrophic and malodorous, but even mild disease can affect the quality of life (4). Mucosal involvement is rare, but oral, oesophageal, vaginal and conjunctival involvement have been described (5–8). The disease is characterized by recurrent exacerbations and remissions lasting months to years (1,3). Both cutaneous infections and UVB exacerbate disease (9–11). Topical corticosteroids with antibacterial agents, either local or systemic, are effective treatments in most patients (1,12).
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Histologically, HHD is characterized by loss of cohesion between keratinocytes (acantholysis) with epidermal clefting or vesiculation. Widespread partial loss of the intercellular bridges between keratinocytes gives the epidermis the appearance of a ‘dilapidated brick wall’ (Fig. 1b). Ultrastructural studies of acantholytic cells in HHD reveal perinuclear aggregates of keratin intermediate filaments that have retracted from desmosomal plaques (13).
The histology, of HHD shows similarities to that of Darier’s disease (DD), but in DD acantholysis is less pronounced, while dyskeratosis is more obvious. Keratinocytes from patients with HHD dissociate spontaneously in organ culture, but acantholysis is suppressed completely by corticosteroids (14–16).
The recent identification of ATP2A2 encoding a sarco- (endo)plasmic reticulum Ca2+ ATPase pump as the defective gene in DD has disclosed a major role of Ca2+ pumps in maintaining cell–cell adhesion and normal differentiation in the epidermis (17). This discovery raised the possibility that another defect in intracellular Ca2+ homeostasis may underlie HHD.
HHD has been linked to chromosomal region 3q21, initially within a 14 cM interval (18). Subsequently linkage studies showed no evidence for locus heterogeneity, and the size of the critical interval was reduced further to a 5 cM region (19,20). One genomic DNA deletion in this region was detected in a single kindred (20). We analyzed additional families, refined the HHD locus to an interval of <3 cM, constructed a physical yeast artificial chromosome(YAC)/bacterial artificial chromosome(BAC)/phage 1-derived artificial chromosome (PAC) map spanning the critical genetic interval and initiated the search for candidate genes.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSNOTE ADDED IN PROOFREFERENCES |
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Genetic refinement of the HHD critical interval
Previous genetic mapping studies indicated that the HHD gene resided within a 5 cM interval defined by the markers D3S1589 and D3S1587. In an attempt to reduce the size of the critical region, we performed genetic analysis on HHD pedigrees with 16 polymorphic DNA markers from this interval. We obtained no evidence for genetic heterogeneity. Critical centromeric meiotic recombinations were found in one family with markers D3S1589, D3S3606 and D3S3607 (Fig. 2), which allowed us to reduce the genetic interval to a 3 cM region. To refine further the critical disease interval, we mapped genetic markers AFMa339wc5 and Mfd-2 between the cluster of markers D3S3584/D3S3606/D3S3607 and marker D3S1587. Both markers show recombination with the disease phenotype in one affected individual (Fig. 2). These results indicate that, based on recombinants, the refined HHD critical region is defined by the flanking markers Mfd-2 and D3S1587. This genetic mapping data guided further positional cloning efforts.
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Physical mapping of the critical region
To develop a physical map of the region containing the HHD locus, 21 markers publicly available and located between D3S1589 and D3S1290 were used to screen the CEPH and ICRF YAC libraries. Thirty-four YACs were identified on the basis of their sequence-tagged site (STS) content. Using the YAC map and its associated markers as a starting framework, we developed a BAC/PAC contig spanning the region from marker D3S3606 to D3S1587 (Fig. 3). The identified BAC/PAC clones were tested for the presence of expressed sequence tags (ESTs) and STSs mapped in the region. Additional markers developed from insert ends of strategically selected BAC/PAC clones were also used for BAC/PAC library screening to isolate new clones. This effort resulted in the construction of a bacterial clone contig between D3S3606 and D3S1587 with two gaps in clone representation (Fig. 3). The map consists of 45 ESTs, nine STSs generated from insert ends of BAC and PAC clones, four further STSs and six polymorphic markers. Mapping of these markers on the contig revealed that AFMa339wc5 was centromeric to Mfd-2, thus defining the HHD critical region between markers Mfd-2 and D3S1587 (Fig. 3).
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Isolation and characterization of cDNA
The identification of ATP2A2 as the defective gene in DD guided our attention to Ca2+-ATPases as candidates for HHD. The EST stSG2295, which is located in the HHD critical interval, shows similarity to calcium transport ATPases. Searching the non-redundant database by blastN with EST stSG2295 identified the partial human mRNA AJ010953 and the alternatively spliced rat homologues (M93017, M93018). A blast search with M93017 against dbEST identified a single human EST AA320285 located upstream of the 5' end of AJ010953. Based on the sequence of EST AA320285, PCR primers were designed to screen a keratinocyte cDNA library. We identified clones 55B17 (from keratinocyte cDNA library RZPD550) and 56D7 (from keratinocyte cDNA library RZPD558), which contain together the full length cDNA (AF189723). Sequence analysis of the isolated cDNA revealed a predicted open reading frame (ORF) of 2712 bp encoding a 903 amino acid protein. The ORF is preceded by 202 bp of 5' untranslated sequence. The sequence around the putative ATG translation start site (ACAATGA) contains the critical –3 purine of a Kozak consensus sequence (21).
By searching the blast dbEST database and sequencing IMAGE clones, alternate polyadenylation was detected. In the 3' untranslated sequence five putative polyadenylation signals (AATAAA) starting at positions 3316, 3719, 3899, 4650 and 4690 are present. Poly(A) tracts starting at positions 4671 and 4712 were identified. The two isolated keratinocyte cDNA clones show an additional alternate polyadenylation at position 3556 using the AGTAAA sequence as a polyadenylation signal. This cDNA sequence shows at least three different sized 3' ends corresponding to UniGene clusters Hs.47168 (EST stSG21646) and Hs.106778. In addition, a splice variant showing an alternative 3' coding and untranslated sequence could be identified by sequence data base analysis. The point of divergence between the two isoforms occurred after codon 892. The alternatively spliced isoform (ATP2C1b) revealed a predicted ORF of 2772 bp encoding a protein of 923 amino acids. Amino acid 893 is in both cases Val, but is encoded by different codons [isoform a (ATP2C1a): GTT; isoform b: GTC]. Isoform b terminates with a 464 bp 3' untranslated sequence containing UniGene cluster Hs.48119 (ESTs stSG2295 and stSG42328). Northern blot hybridization of total keratinocyte RNA using a probe specific for the 3'UTR of ATP2C1 showed a 4.5 kb transcript highly expressed (data not shown).
Genomic organization of ATP2C1
The EST stSG2295 mapped onto BAC clones RG139I22, RG356K14 and RG148L10. To obtain the exon–intron organization of the ATP2C1 gene we performed vectorette-PCR with primers generated from the cDNA sequence. During this procedure it became obvious that BAC RG356K14 contains only the 3' end of the ATP2C1 gene, whereas clone RG139I22 harbors the complete gene. By sequencing vectorette-PCR products we obtained 30 kb of novel sequence and were able to predict all exon–intron boundaries of ATP2C1 based on sequence comparison between the cDNA and the vectorette-PCR product sequences (Table 1). The ORFs are distributed across 26 and 27 exons, respectively, starting in exon 2; these range in size from 36 to 172 bp (excluding the 3' exons). Exon 1 is highly GC-rich (76.3 %), contains 20 CG dinucleotides and forms with the adjacent sequence a putative 1 kb CpG island. The GT dinucleotide at position 2879–2880 of the cDNA of isoform a is used as a cryptic donor splice site in isoform b eliminating the last 33 bp of the predicted ORF and the complete 3' untranslated sequence of exon 27. This sequence is replaced by exon 28 in isoform b.
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In conclusion, we identified ATP2C1, which is present as two splice variants with at least four different 3' untranslated termini. Analysis of 30 kb sequence obtained by sequencing vectorette-PCR products showed that at least four UniGene clusters are assigned to this gene.
Computational analysis of ATP2C1 and its encoded protein
The predicted protein contains all of the conserved domains commonly found in P-type ion-transport ATPases. The seven amino acid motif DKTGT-[LIVMS]-[TIS] (motif no. PS00154 in the PROSITE (22) database of protein motifs), which classifies a protein as a P-type ATPase, next to the aspartate, which is phosphorylated during the reaction cycle, is found in the sequence DKTGTLT (position 334–340) in the protein encoded by ATP2C1. The enzyme also contains two sequences around the reactive Lys-464 (23) and Asp-628 (24), which are thought to form part of the ATP binding site. All three amino acid residues have been reported to be essential for the activity of ion-transporting ATPases.
Similarity comparisons demonstrate that the amino acid sequence encoded by ATP2C1 most closely resembles those of rat SPLA Ca2+ pump (25), the yeast secretory pathway PMR1 pump (26) and the sarco(endo)plasmic calcium ATP-ase (SERCA) (27) and plasma membrane calcium ATP-ase (PMCA) classes of mammalian Ca2+ pumps (28). A multiple-sequence alignment of the predicted amino acid sequence of the product encoded by ATP2C1 with SPLA, PMR1, SERCA2 and PMCA2 (Fig. 4) reveals a high number of conserved positions including sequences around Asp-334, Lys-464 and Asp-628. The protein has the four major hydrophobic domains in the N-terminal half of the protein and the six hydrophobic domains in the C-terminal half that correspond to the known transmembrane domains of the other transport ATPases. The highest degree of similarity occurs between the predicted transmembrane domains 2 and 3, and between the transmembrane domains 4 and 5. By analogy with other P-type ATPases, these conserved regions are predicted to contain the cytoplasmic domains involved in ATP binding, phosphorylation and conformational changes of the enzyme. Thus, the predicted protein has the same apparent transmembrane organization and comprises all of the conserved domains characteristic of the P-type ATPases. In addition, the protein contains six potential Ca2+ binding sites, three of which (E292, N722 and D726) are located in transmembrane domains 4 and 6, and are conserved among all five pumps (29), while three other sites (A697, M725 and D803) are located in transmembrane domains 5, 6 and 8, respectively, and are conserved among ATP2C1, SPLA and PMR1 (Fig. 4).
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In pairwise comparisons, the predicted protein exhibits 97% amino acid identity to rat SPLA, 49% identity to yeast PMR1, 37% identity to human SERCA2 and 31% identity to human PMCA2. It should be noted that both the rat SPLA and yeast PMR1 pumps are medial-Golgi Ca2+ pumps that exhibit 50% amino acid identity, while SERCA2 and PMCA2, which are members of two different classes of human Ca2+ pumps, share only 29% amino acid identity. On the basis of these comparisons, it seems likely that the protein encoded by ATP2C1 represents a third class of human Ca2+ ATPase pump, which is the human homologue for the rat SPLA and the yeast PMR1 pump.
ATP2C1 mutations in HHD families
ATP2C1 cDNA was amplified from lymphoblasts from seven patients from different HHD families and was screened for mutations by Conformation Sensitive Gel Electrophoresis (CSGE) analysis. The entire coding sequence and flanking intron boundaries were amplified from genomic DNA in nine additional HHD patients from unrelated families. Bands of abnormal electrophoretic mobility were seen in 14 patients and sequencing of these PCR products revealed 13 different mutations (Table 2, Fig. 5). No mutation was found in two families with typical HHD after direct sequencing of the entire coding region of ATP2C1 cDNA. Four substitutions resulted in premature termination codons (PTCs) (R39X, Q427X, S546X, Q849X). Two deletions (2180delG and 2326delTTTG) created shifts in the reading frame and resulted in PTCs. Three splice site mutations occurred at the guanine of the conserved AG dinucleotide in the acceptor splice site of introns 11, 18 and 26. These mutations probably alter splicing of exons 12, 19 and 27, respectively, although the precise consequence is unknown as RNA was not available for these patients. Three missense mutations were identified, P185L, C328Y and T554I, which are located in the predicted cytoplasmic ß-strand, phosphorylation and nucleotide binding domains of the molecule, respectively. T554I was present in two families not known to be related (Table 2). Each of these mutations result in non-conservative amino acid substitutions at positions conserved with the rat SPLA sequence and the human ATP2A2b sequence (Fig. 4). None of these mutations were detected in 50 control individuals (100 chromosomes), indicating that these mutations are not likely to be neutral polymorphisms. Finally, a guanine deletion followed by a large insertion of ~23 adenines and a thymine was identified in family NC2. Sequencing of 12 subclones of this region revealed the number of adenines inserted to vary from 19 to 26, making the precise consequence of this mutation difficult to predict.
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During the search for mutations, a G2550A substitution in exon 26 of ATP2C1 was found. This substitution does not change the amino acid coding and does not alter transcript size in the relevant patients, so is presumed to be a neutral polymorphism. Probands from families OX8, FR1, NC1, OX6 and FR2 were found to be heterozygous for this polymorphism; the remaining probands were all homozygous for the guanine at this site.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSNOTE ADDED IN PROOFREFERENCES |
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We used a positional cloning strategy to identify ATP2C1, the defective gene in HHD. We refined the genetic interval to <1 cM by identifying critical meiotic recombinants, developed a physical map of the region and identified a new Ca2+-ATPase using a combination of bioinformatic analysis and keratinocyte cDNA library screening. We identified 13 mutations pre- dicted to alter the ATP2C1 gene product in affected members of 14 HHD families, thus demonstrating that ATP2C1 is the gene involved in HHD.
The protein is a new member of the P-type ion-transport ATPases, which is highly expressed in keratinocytes. Multiple sequence alignment suggests that it is the human homologue for the rat SPLA and the yeast PMR1 Ca2+ pumps, both of which are medial-Golgi pumps (25,26,30). The conservation of functional domains that are common to other Ca2+-ATPases suggests that ATP2C1 actively transports Ca2+ from the cytosol to the lumen of the Golgi apparatus. ATP2C1 would thus play an important role in maintaining Ca2+ concentrations within compartments of the Golgi apparatus. Our results provide convincing evidence that disruption of ATP2C1 causes HHD.
Six of these mutations are predicted to prevent expression of the corresponding protein to produce a truncated or absent protein. In three families, the mutation alters a predicted splice site. Three other mutations are predicted to cause missense changes that are unlikely to be polymorphisms as they were not found in 50 controls. These mutations are located in predicted cytoplasmic domains of the molecule thought to be involved in catalytic activities of P-type ATPases. Mutation P185L occurred in the ß-strand domain at a position that is conserved between ATP2C1, rat SPLA and human ATP2A2. Mutation C328Y is located in the phosphorylation domain, five amino acids upstream of the conserved phosphorylation sequence DKTGT, which is found in all P-type ATPases. Mutation T554I is located in the nucleotide binding site of the molecule and occurs at a position that is conserved among the three types of Ca2+ pumps (Fig. 4). This mutation is identical in two families (OX1 and OX2) of British origin, and studies are under way to determine if this reflects a founder effect. Although the precise consequence of these missense mutations could not be predicted, the expected effect of the other mutations is the absence of production of the mutated polypeptide. These findings support a mechanism through haploinsufficiency, suggesting that epidermal cells are sensitive to ATP2C1 gene dosage. Interestingly, a similar disease mechanism has been proposed for the majority of ATP2A2 mutations found in patients with DD (17,31,32). Although both diseases show acantholysis of epidermal cells and may show some clinical overlap, erosions and blistering are prominent features in HHD while dyskeratosis predominates in patients with DD. Thus, dysfunction of ATP2A2 and ATP2C1 appears to have different effects on cell–cell adhesion and differentiation of the epidermis.
Extracellular calcium plays a crucial role in regulating differentiation and adhesion of cultured keratinocytes (33,34). Low levels of Ca2+ induce their proliferation while physiological levels of extracellular Ca2+ induce cell-to-cell adhesion and keratinocyte differentiation through the expression of many markers associated with formation of a cornified layer. The mechanisms that are implicated are unclear, but probably involve changes in intracellular calcium levels. In normal human epidermis, the presence of an increasing Ca2+ gradient from the basal cells to the upper granular cells (35) where several Ca2+-mediated differentiation markers are expressed, strongly suggests that Ca2+ also plays an important role in vivo. Our results, together with the recent identification of ATP2A2 mutations in DD, indicate that Ca2+ stores in the endoplasmic reticulum and the Golgi apparatus play a key role in epidermal differentiation and/or adhesion. Reduced ATP2C1 activity is thought to decrease intraluminal Ca2+ concentrations in the Golgi and may also have an effect on cytosolic Ca2+ concentration. Among processes carried out in the lumen of the Golgi, post-translational modifications such as glycosylation, folding, trafficking and/or sorting of key molecules involved in cell-to-cell adhesion may be altered (36,37). By analogy with Pmr1-defective yeast, alterations in the secretory pathway and/or the clearance of misfolded proteins could also be involved (38,39). It is also possible that dysfunction of the ATP2C1 pump could have an effect on cytosolic Ca2+ concentrations, thus impairing intracellular signaling pathways that regulate cell adhesion through the phosphorylation of target proteins and/or the regulation of gene transcription (40–42).
Further studies on the precise effects of ATP2C1 mutations on cell-to-cell adhesion and differentiation through the identification of target molecules should improve our understanding of the Ca2+-dependent biological pathway involved in the disease. This may help in the development of specific therapeutic approaches aiming to restore normal Ca2+ homeostasis in the epidermis of these patients.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSNOTE ADDED IN PROOFREFERENCES |
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Families
A total of 149 individuals, including 69 affected members from 15 families were included in this study. Diagnosis was established by a dermatologist, on the basis of physical examination and was confirmed by histological examination of a skin biopsy in at least one member of each family. Peripheral blood was collected in EDTA for DNA isolation, and in acid citrate dextrose for the establishment of Epstein–Barr virus-transformed lymphocytes by standard methods. In addition, genomic DNA was extracted from buccal smears. Blood samples, buccal smears and skin biopsies were obtained after patients had given informed consent.
Genotyping
Sixteen polymorphic DNA markers ( D3S1589, AFM203wc10, D3S3584, D3S3606, D3S3607, AFMa339wc5, Mfd-2, D3S1587, D3S3548, D3S3514, D3S1292, D3S1596, D3S1273, D3S1290, D3S1541, ACPP) between D3S1589 and D3S1290 were used in this study. Forward PCR primers were labelled with either 6-FAM, HEX or TET phosphoramidite. Microsatellite analyses were performed by PCR amplification of 40 ng of genomic DNA in a 25 µl reaction containing 10 pmol each of the forward and reverse primer, 200 µM dNTPs, 1x reaction buffer (1.5 mM MgCl2, 45 mM Tris pH 8.8, 11 mM [NH4]SO4 pH 8.8, 6.7 mM ß-mercaptoethanol and 4.5 µM EDTA), and 0.7 U of AmpliTaq DNA polymerase (Perkin Elmer Applied Biosystems, Foster City, CA). Amplification was performed with an initial denaturation at 94°C for 5 min and 35 cycles at 94°C for 30 s, at either 50, 55 or 58°C for 30 s, and at 72°C for 30 s, in an MJ Research PTC-225 thermal cycler. Products were mixed with an equal amount of formamide buffer, and run on a 373A sequencer (Applied Biosystems), followed by analysis with GENESCAN (version 2.0.2) and GENOTYPER (version 1.1) software to derive allele sizes.
Clone isolation
YACs were identified from total human genomic CEPH Mega-YAC and ICRF YAC libraries. PACs were identified from the RPCI1, 3–5 library created by P. de Jong, and BACs were identified either from the Research Genetics human BAC library or from the CEPH BAC library. Clones were identified by PCR-based screening of DNA pools with publicly available STSs. After isolation, individual clones were tested by colony PCR to establish their STS content. As the clone contig assembly proceeded, clones were selected for the generation of insert end-specific STSs by vectorette-PCR.
Development of YAC-, BAC- and PAC-insert end-specific STSs
YAC-, BAC- and PAC-end sequences were isolated by established vectorette-mediated PCR techniques (43,44). DNA was digested with RsaI, AluI, HaeIII, PvuII, HpaI, EcoRV and ScaI and was blunt-end ligated to double-stranded vectorette. The vectorette libraries were amplified with both the vectorette primer and a primer for either the right or the left arm of pYAC4, for YAC insert end recovery, and were amplified with either T7 or Sp6 primers for isolation of BAC and PAC insert ends. End fragments were sequenced, and PCR primers were designed.
Analysis of BAC and PAC clones
Pulsed-field gel electrophoresis separation of NotI-digested BAC and PAC clones was used to determine the insert size, as well as the presence of internal NotI restriction sites. The digested samples were run on 1% agarose gels at 6 V/cm for 20 h, at an initial pulse time of 4 s and a final pulse time of 20 s, with a CHEF DRII (Bio-Rad, Hercules, CA) apparatus.
FISH analysis
Clones were tested by FISH analysis for chromosomal location and chimerism. One microgram of DNA was biotinylated by nick translation with the Bionick Labelling System (Life Technologies, Rockville, MD) and was processed by standard techniques.
Mutation analysis
Total RNA was extracted from immortalized lymphocyte cell lines using Trizol (Life Technologies). First strand cDNA was synthesized from RNA (5 µg) with Superscript reverse transcriptase (Life Technologies) and random hexamer primers (Pharmacia Biotech., Uppsala, Sweden). The entire coding sequence of ATP2C1 cDNA was amplified using overlapping sets of primers (sequence available on request). PCRs were carried out with standard reaction mixes containing 2.5 mM MgCl2. After an initial denaturation step at 94°C for 15 min, 35 cycles of amplification consisting of 30 s at 94°C, 30 s at 58°C and 30 s at 72°C were performed. RT–PCR products were run on 3% agarose gels for detection of splicing abnormalities prior to mutation screening by CSGE. For CSGE analysis, the formation of heteroduplexes was enhanced by heating RT–PCR products to 98°C for 5 min, followed by 68°C for 1 h. Samples (5 µg) were loaded onto a gel containing 10% polyacrylamide, 15% formamide, 10% ethylene glycol and 0.5x glycerol-tolerant buffer (USB) and were subjected to electrophoresis at 500 V for 16 h. The gel was stained with ethidium bromide and visualized under an ultraviolet transilluminator. RT–PCR fragments showing aberrant migration patterns or extra bands were sequenced in forward and reverse orientations. PCR products showing aberrant migration patterns or extra bands were purified using a QIAquick PCR purification kit (Qiagen). These were then directly sequenced in both directions by means of the BigDye Terminator Cycle Sequencing Ready Reaction kit (PE-Applied Biosystems) and were run on an ABI 377 DNA Sequencer (PE-Applied Biosystems). Where RNA was not available, all 26 translated exons plus flanking splice sites were amplified from genomic DNA (primer sequences available on request), analysed by CSGE and sequenced as described above. The different-sized products resulting from amplification of exon 2 in the proband of family NC2 were subcloned into pGEM-T vector (Promega Corp., Madison, WI) and multiple subclones were sequenced. The presence of the mutations was verified by restriction digestion or denaturing high-performance liquid chromatography.
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ACKNOWLEDGEMENTS |
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We are grateful to the patients and their families. This work was supported by The Wellcome Trust. R.S. was supported by the EU HCMP fellowship CT940570 and the Deutsches Human Genome Project (DHGP/BMBF Grant 01KW9608). A.H. held a DEBRA UK fellowship and is a Wellcome Trust Senior Clinical Fellow. A.P.M. is a Wellcome Principal Research Fellow. The authors would like to thank the DHGP for providing genomic and cDNA clones.
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NOTE ADDED IN PROOF |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSNOTE ADDED IN PROOFREFERENCES |
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Whilst this manuscript was under review, Z. Hu et al. reported the identification of ATP2C1 as the HHD gene [mutations in ATP2C1, encoding a calcium pump, cause Hailey–Hailey disease, Nature Genet., (2000) 24, 61–65]. In this article, the coding sequence of ATP2C1 starts 16 amino acids upstream of the sequence which we describe. Genomic sequencing showed that these two isoforms result from alternative splicing of an additional exon located between exons 1 and 2 (data not shown). Alternative splicing also accounts for ATP2C1b described by Z. Hu et al., which results from joining exon 26 to exon 28.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +44 1865 287 502; Fax: +44 1865 287 650; Email: anthony.monaco@well.ox.ac.uk
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSNOTE ADDED IN PROOFREFERENCES |
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