Identification and molecular characterization of the first complete genome sequence of Human Parechovirus type 15.

Using a metagenomics approach, we have determined the first full-length genome sequence of a human parechovirus type 15 (HPeV15) strain, isolated from a child with acute flaccid paralysis and co-infected with EV-A71. HPeV15 is a rarely reported type. To date, no full-length genome sequence of HPeV15 is available in the GenBank database, where only limited VP1 sequences of this virus are available. Pairwise comparisons of the complete VP1 nucleotide and deduced amino acid sequences revealed that the study strain belongs to type 15 as it displayed 79.6% nucleotide and 93.4% amino acid identity with the HPeV15 prototype strain. Comparative analysis of available genomic regions and phylogenetic analysis using the P2 and P3 coding regions revealed low nucleotide identity to HPeV reference genomes. Phylogenetic and similarity plot analyses showed that genomic recombination events might have occurred in the UTRs and nonstructural region during HPeV15 evolution. The study strain has high similarity features with different variants of HPeV3 suggesting intertypic recombination. Our data contributes to the scarce data available on HPeVs in Africa and provides valuable information for future studies that aim to understand the evolutionary history, molecular epidemiology or biological and pathogenic properties of HPeV15.

Association of a single nucleotide polymorphism in the ubxn6 gene with long-term non-progression phenotype in HIV-positive individuals.

OBJECTIVES: The long-term non-progressors (LTNPs) are a heterogeneous group of HIV-positive individuals characterized by their ability to maintain high CD4+ T-cell counts and partially control viral replication for years in the absence of antiretroviral therapy. The present study aims to identify host single nucleotide polymorphisms (SNPs) associated with non-progression in a cohort of 352 individuals. METHODS: DNA microarrays and exome sequencing were used for genotyping about 240 000 functional polymorphisms throughout more than 20 000 human genes. The allele frequencies of 85 LTNPs were compared with a control population. SNPs associated with LTNPs were confirmed in a population of typical progressors. Functional analyses in the affected gene were carried out through knockdown experiments in HeLa-P4, macrophages and dendritic cells. RESULTS: Several SNPs located within the major histocompatibility complex region previously related to LTNPs were confirmed in this new cohort. The SNP rs1127888 (UBXN6) surpassed the statistical significance of these markers after Bonferroni correction (q = 2.11 × 10-6). An uncommon allelic frequency of rs1127888 among LTNPs was confirmed by comparison with typical progressors and other publicly available populations. UBXN6 knockdown experiments caused an increase in CAV1 expression and its accumulation in the plasma membrane. In vitro infection of different cell types with HIV-1 replication-competent recombinant viruses caused a reduction of the viral replication capacity compared with their corresponding wild-type cells expressing UBXN6. CONCLUSIONS: A higher prevalence of Ala31Thr in UBXN6 was found among LTNPs within its N-terminal region, which is crucial for UBXN6/VCP protein complex formation. UBXN6 knockdown affected CAV1 turnover and HIV-1 replication capacity.

Are we modelling the correct dataset? Minimizing false predictions for dengue fever in Thailand.

Models describing dengue epidemics are parametrized on disease incidence data and therefore high-quality data are essential. For Thailand, two different sources of long-term dengue data are available, the hard copy data from 1980 to 2005, where hospital admission cases were notified, and the electronic files, from 2003 to the present, where clinically classified forms of disease, i.e. dengue fever, dengue haemorrhagic fever, and dengue shock syndrome, are notified using separate files. The official dengue notification data, provided by the Bureau of Epidemiology, Ministry of Public Health in Thailand, were cross-checked with dengue data used in recent publications, where an inexact continuous time-series was observed to be consistently used since 2003, affecting considerably the model dynamics and its correct application. In this paper, numerical analysis and simulation techniques giving insights on predictability are performed to show the effects of model parametrization by using different datasets.

Genetic diversity in human erythrocyte pyruvate kinase.

Previously, we have shown that pyruvate kinase, liver and red cell isoform (PKLR) deficiency protects mice in vivo against blood-stage malaria, and observed that reduced PKLR function protects human erythrocytes against Plasmodium falciparum replication ex vivo. Here, we have sequenced the human PKLR gene in 387 individuals from malaria-endemic and other regions in order to assess genetic variability in different geographical regions and ethnic groups. Rich genetic diversity was detected in PKLR, including 59 single-nucleotide polymorphisms and several loss-of-function variants (frequency 1.5%). Haplotype distribution and allele frequency varied considerably with geography. Neutrality testing suggested positive selection of the genein the sub-Saharan African and Pakistan populations. It is possible that such positive selection involves the malarial parasite.

Linkage disequilibrium of polymorphic RAET1 genes in Thais.

Retinoic acid early transcripts-1 (RAET1) or unique long 16 (UL-16) binding proteins (ULBPs) is a gene cluster encoding for molecules acting as ligands to natural killer group 2 D (NKG2D), a receptor expressed on immune cells. Binding of these ligands to the receptor activates immune cells leading to killing of tumor cells and also viral-infected cells. The information on polymorphism of RAET1 is limited. In this report, we analyze the linkages between four polymorphic RAET1 genes: RAET1E, RAET1G, RAET1H and RAET1L, in 318 unrelated Thais. The strongest linkage disequilibrium was found between RAET1E and RAET1G, with P-value, D' and r(2) of <5.0 x 10(-5), 0.707 and 0.840, respectively. RAET1E(*)001 was found to be in linkage disequilibrium with RAET1G(*)002, and RAET1E(*)002 with RAET1G(*)001. Evidently, there were possible RAET1 haplotypes with haplotype frequencies of more than 10% consisting of RAET1E(*)001; RAET1G(*)002; RAET1H(*)001; RAET1L(*)001 and RAET1E(*)002; RAET1G(*)001; RAET1H(*)002; RAET1L(*)003. This study provides basic information on polymorphisms of RAET1 and possible RAET1 haplotypes in Thais.

Genetic study of ICAM1 in clinical malaria in Senegal.

Many genes have been implicated in the risk of severe malaria, generally based on candidate gene studies in case/control populations. Among these genes, there has been conflicting reports for the implication of a variant of the intercellular adhesion molecule 1 (ICAM1), ICAM1(Kilifi), in the risk of severe malaria, while in vitro studies provided independent support for a functional role of this variant. In order to explore the possible implication of ICAM1 in the susceptibility/resistance to malaria and to try to understand its clinical relevance in the disease process, we have conducted linkage and association studies of ICAM1 in two Senegalese villages located in regions of endemic malaria. We explored the full genetic variability of ICAM1, and tested it on several clinical malarial traits which are under genetic control, focusing principally on variables related to the parasite density and the number of malarial attacks. Our study provides no evidence for a role of ICAM1 variability on the malarial phenotypes studied.

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.

Mosaicism for ATP2A2 mutations causes segmental Darier's disease.

Epidermal naevi are localized malformations of the epidermis consisting of verrucoid scaly papules and plaques following Blaschko's lines. Genetic mosaicism has been proposed to underlie the development of linear epidermal naevi. Rarely, epidermal naevi show acantholytic histology similar to Darier's disease, a dominantly inherited skin condition characterized by widespread warty papules. As patients with acantholytic dyskeratotic naevi often give a history of worsening after sun exposure and the lesions are typical of Darier's disease, numerous authors have proposed that these patients have segmental Darier's disease. The postulated relationship has not been proven, however. Recently, we identified ATP2A2, which encodes the sarco/endoplasmic reticulum Ca(2+) ATPase isoform 2 as the defective gene in Darier's disease. In this report, we investigated the involvement of ATP2A2 in acantholytic dyskeratotic naevi following Blaschko's lines in two patients. We identified a nonsense mutation (Y894X) in the first patient and a nonconservative glycine to arginine mutation at codon 769 (G769R) in the other patient. These mutations were present in affected skin, and were not detected in unaffected skin or in leukocytes. We conclude that acantholytic dyskeratotic naevi can arise from a somatic mutation in ATP2A2. These individuals are mosaics for the mutation, but the risk of transmission of generalized Darier's disease will depend on whether the germline is affected. Our findings provide further evidence that Blaschko's lines do reflect genetic mosaicism and that the term acantholytic dyskeratotic naevus might be replaced in the future by segmental Darier's disease induced by postzygotic mosaicism. J Invest Dermatol 115:1144-1147 2000

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.

Mutations in ATP2A2, encoding a Ca2+ pump, cause Darier disease.

Darier disease (DD) is an autosomal-dominant skin disorder characterized by loss of adhesion between epidermal cells (acantholysis) and abnormal keratinization. Recently we constructed a 2.4-Mb, P1-derived artificial chromosome contig spanning the DD candidate region on chromosome 12q23-24.1. After screening several genes that mapped to this region, we identified mutations in the ATP2A2 gene, which encodes the sarco/endoplasmic reticulum Ca2(+)-ATPase type 2 isoform (SERCA2) and is highly expressed in keratinocytes. Thirteen mutations were identified, including frameshift deletions, in-frame deletions or insertions, splice-site mutations and non-conservative missense mutations in functional domains. Our results demonstrate that mutations in ATP2A2 cause DD and disclose a role for this pump in a Ca(2+)-signalling pathway regulating cell-to-cell adhesion and differentiation of the epidermis.

Deletions within COL7A1 exons distant from consensus splice sites alter splicing and produce shortened polypeptides in dominant dystrophic epidermolysis bullosa.

We describe two familial cases of dominant dystrophic epidermolysis bullosa (DDEB) that are heterozygous for deletions in COL7A1 that alter splicing, despite intact consensus splice-site sequences. One patient shows a 28-bp genomic deletion (6081del28) in exon 73 associated with the activation of a cryptic donor splice site within this exon; the combination of both defects restores the phase and replaces the last 11 Gly-X-Y repeats of exon 73 by a noncollagenous sequence, Glu-Ser-Leu. The second patient demonstrates a 27-bp deletion in exon 87 (6847del27), causing in-frame skipping of this exon; consensus splice sites, putative branch sites, and introns flanking exons 73 and 87 showed a normal sequence. Keratinocytes from the probands synthesized normal and shortened type VII collagen polypeptides and showed intracellular accumulation of type VII procollagen molecules. This first report of genomic deletions in COL7A1 in DDEB suggests a role for exonic sequences in the control of splicing of COL7A1 pre-mRNA and provides evidence that shortened type VII collagen polypeptides can alter, in a dominant manner, anchoring-fibril formation and can cause DDEB of differing severity.

Refined genetic mapping of the darier locus to a <1-cM region of chromosome 12q24.1, and construction of a complete, high-resolution P1 artificial chromosome/bacterial artificial chromosome contig of the critical region.

Darier disease (DD) (MIM 124200) is an autosomal dominant skin disorder characterized by loss of adhesion between epidermal cells and by abnormal keratinization. We present linkage analysis showing, in four families, key recombination events that refine the location of the DD locus on chromosome 12q23-24.1 to a region of <1 cM. We have constructed a YAC/P1 artificial chromosome (PAC)/bacterial artificial chromosome (BAC)-based physical map that encompasses this refined DD region. The map consists of 35 YAC, 69 PAC, 16 BAC, and 2 cosmid clones that were ordered by mapping 54 anonymous sequence-tagged sites. The critical region is estimated to be 2.4 Mb in size, with an average marker resolution of 37.5 kb. The refinement of the critical interval excludes the ALDH2, RPL6, PTPN11, and OAS genes, as well as seven expressed sequence tags (ESTs) previously mapped in the DD region. The three known genes (ATP2A2, PPP1CC, and SCA2) and the 10 ESTs mapped within the critical region are not obvious candidates for the DD gene. Therefore, this detailed integrated physical, genetic, and partial transcript map provides an important resource for the isolation of the DD gene and, possibly, other disease genes.

A sequence-ready physical map of a region of 12q24.1.

We developed a sequence-ready map of a part of human chromosome 12q24.1. We utilized a number of sequence-tagged site (STS) markers from 12q24.1 to screen large insert bacterial chromosome libraries and a chromosome 12-specific cosmid library. The clones were assembled into contiguous sets (contigs) by STS-content analysis. Contigs were extended by obtaining end sequences of bacterial clones, generation of additional STSs, rescreening the libraries, and screening the additional clones for the presence of STSs. The resulting contig covers nearly 2 Mb of DNA and provides an average marker resolution of 16 kb. Based on the STS content, we developed fingerprints of a subset of clones. The STS content and fingerprint data allowed us to define a minimal tiling path of clones. These clones are being used to sequence this part of chromosome 12. This contig contains the Ataxin 2 gene, and it covers the interval harboring the gene responsible for Darier disease.

Comparison of psoralen-UVB and psoralen-UVA photochemotherapy in the treatment of psoriasis.

BACKGROUND: PUVA treatment of psoriasis is usually given with broad-band fluorescent UVA lamps. Narrow-band UVB exposure after oral methoxsalen has been shown to achieve a greater therapeutic response in psoriasis than identical UVB exposure given without psoralen. OBJECTIVE: The purpose of this study was to compare conventional PUVA with psoralen-UVB therapy in psoriasis. METHODS: We studied 100 patients with plaque-type psoriasis who were randomly selected to receive either conventional psoralen-UVA or psoralen-UVB treatment. RESULTS: No significant difference was found between the two treatments in the proportion of patients whose skin cleared during treatment or in the number of exposures required for clearance of psoriasis. As expected, the cumulative UV dose for clearance was smaller in the group treated with UVB compared with those receiving UVA. Side effects and disease status at 3 months after the end of treatment were similar for the two groups. CONCLUSION: Psoralen-UVB treatment of psoriasis is as effective as conventional PUVA. The mechanism of psoralen-311 nm UVB action on psoriasis requires study to predict the long-term safety of this treatment.

Telomerase activity in oral leukoplakia and head and neck squamous cell carcinoma.

The expression of telomerase, a ribonucleoprotein complex, is necessary to overcome cellular senescence, and it is associated with immortal cells and cancer. However, its role in precancerous lesions such as oral leukoplakias is less known. The purpose of this study is to investigate the presence of telomerase activity in oral leukoplakia and the relationship between the enzyme and multistep tumorigenesis. Telomerase activity was detectable in 14 of 16 human head and neck squamous cell carcinomas and 10 of 26 oral leukoplakia tissues. We also showed that the expression of telomerase in the premalignant lesions was associated with phenotypic progression, the degree of dysplasia. These results indicate that telomerase is activated frequently during the late stage of oral premalignancy and may play a crucial role in head and neck squamous cell carcinogenesis.

Calculation of 8-methoxypsoralen dose according to body surface area in PUVA treatment.

In 41 patients about to start PUVA, the dose of 8-methoxypsoralen (8-MOP) was calculated conventionally according to body weight (0.6 mg/kg), or according to body surface area (25 mg/m2) predicted from height and weight measurements. The two different methods of dosing were used on consecutive treatment days and the plasma 8-MOP concentration was measured on each occasion 2 h after ingestion of the crystalline form of 8-MOP, given to the nearest 10 mg. Body weight calculated doses ranged from 30 to 60 mg with a significant difference in the plasma 8-MOP concentration between the dose groups, indicating a systematic variation according to the weight of the patient. When calculated according to body surface area, only two doses were used (40 or 50 mg), and there was no significant difference in plasma 8-MOP concentration between the groups. Calculation of the dose of 8-MOP using body surface area may be performed quickly and simply provided the height and weight of individual patients is known. We provide evidence that this method of dosing will improve the therapeutic effect of PUVA in psoriasis.

Etiology of erythema nodosum.

One hundred patients with biopsy-proven erythema nodosum were studied at Ramathibodi Hospital from 1982 to 1992 to find out the etiology of this disease. Eighty-eight were females while twelve were males, with an age range from 6 to 72 years old (mean, 31 years old). Abnormal laboratory findings in these patients included elevation of erythrocyte sedimentation rate (76.9%), increase anti-streptolysin-O titer (10.7%), abnormal chest roentgenogram (16.7%), positive tuberculin test (50%). The cause of erythema nodosum is still unknown in a large group of patients, and it was found only in twenty-eight patients (28%). Twelve patients had tuberculosis, seven had history of antibiotic administration, six probably had streptococcal infection and the other three had Behcet's disease.

Effect of aspirin and nonsteroidal antiinflammatory drug therapy on bleeding complications in dermatologic surgical patients.

BACKGROUND: Aspirin and nonsteroidal antiinflammatory drugs (NSAIDs) inhibit platelet cyclooxygenase activity, resulting in altered platelet function and thus potentially enhanced bleeding. OBJECTIVE: We examined the frequency of operative bleeding complications in dermatologic surgical patients taking these drugs and the value of template bleeding time estimates in predicting this complication. METHODS: Bleeding time was measured with and without therapy in 23 patients and was correlated to bleeding complications after skin tumor or benign lesion excision in 40 patients taking aspirin, 21 taking NSAIDs, and 20 taking neither drug. RESULTS: Bleeding time dropped significantly (p < 0.01) when patients stopped therapy for at least 5 days (median, 7 days), although bleeding time was prolonged in only 6 of 16 patients taking aspirin and 2 of 7 taking NSAID. In patients who continued antiplatelet drugs during surgery, bleeding time was prolonged in 8 of 40 patients taking aspirin and in 1 of 21 treated with NSAIDs. Excessive intraoperative bleeding occurred in three aspirin-treated patients, all of whom had a prolonged bleeding time, compared with none of those with normal bleeding times (p < 0.001, Fisher's exact probability test) and with none of those taking NSAIDs. Postoperative ooze requiring a dressing replacement occurred in one NSAID-treated patient and in three patients taking neither drug. CONCLUSION: Bleeding time is increased by aspirin and NSAID therapy but is prolonged beyond the normal range in only approximately 25% of aspirin-treated and 10% of NSAID-treated patients. Intraoperative bleeding complications occurred only in patients receiving aspirin who had a prolonged bleeding time. Postoperative oozing occurred only in NSAID-treated and in untreated patients and thus is probably unrelated to antiplatelet therapy. Patients with a normal bleeding time can continue aspirin or NSAID therapy before dermatologic surgery.

Prevalence of thyroid diseases in patients with alopecia areata.

BACKGROUND: The prevalence of thyroid disease in patients with alopecia areata previously reported varied from 0 to 28%. These thyroid diseases, include Hashimoto's thyroiditis, Graves' disease, simple goiter, and others. METHODS: The prevalence of thyroid diseases was determined in 152 consecutive patients with alopecia areata who presented to the dermatology clinic. A complete history was taken and a physical examination was performed. Thyroxine, triiodothyronine, thyroid-stimulating hormone, and microsomal antibody levels were measured in every patient. The control group consisted of 152 age- and sex-matched volunteers who had skin diseases other than alopecia areata or autoimmune disorders. RESULTS: Among 152 patients, age 10-59 years, four cases (2.6%) had a small simple goiter. Microsomal antibodies were detected in seven other patients (4.6%) with titers ranging from 1:100 to 1:1600. None of these seven patients had signs or symptoms of thyroid disease. Five cases (3.3%) of the control group had positive microsomal antibody tests with titers ranging from 1:100 to 1:400. The prevalence of positive microsomal antibodies in the alopecia areata group was not statistically different from the control group (chi 2 = 0.347, DF = 1, P = 0.5558). CONCLUSIONS: Among 152 patients with alopecia areata, 4.6% of patients had microsomal antibodies and 2.6% had a small simple goiter. Thus the prevalence of thyroid disease among these patients was 7.2%. The prevalence of positive microsomal antibodies in 4.6% of the patients was not statistically different from that of the control group.