Linear allele-specific long-range amplification: a novel method of long-range molecular haplotyping.

Haplotypes have been repeatedly shown to be more powerful than collections of single-locus markers in gene-mapping studies. Various haplotyping methods including statistical estimation are employed but molecular haplotyping, the acquisition of information directly on physical DNA sequences, has been in demand for its accuracy and independence from family pedigrees. We investigated the allelic specificity of long-range PCR, which was successful for long-range haplotyping in recent reports, and found problems of initial mispriming and crossover amplification significantly confounded its application. Based on these observations, we designed a novel method based on linear amplification of a hemizygous DNA segment with a single phosphorothioate-modified oligonucleotide. Our results revealed, with a single nucleotide polymorphism as the discriminative marker, downstream haplotypes of 14-15 kb DNA segment could be confidently scored. With two rounds of the method and five single nucleotide polymorphisms, molecular haplotypes of 29.3 kb spanning the HCR and CDSN genes, two genes associated with the susceptibility of psoriasis, of 11 members, belonging to a CEPH family, were revealed. Clear Mendelian segregation of 35 highly heterozygous SNPs confirmed the accuracy of the method. Problems of low specificity associated with long-range PCR were not observed. The simplicity, along with long-sequence accessibility and feasibility of a single nucleotide difference as the discriminative marker indicated our method holds promise for future gene-mapping studies.

Touchdown thermocycling program enables a robust single nucleotide polymorphism typing method based on allele-specific real-time polymerase chain reaction.

Different methods have been developed for single nucleotide polymorphism (SNP) typing during recent years. Allele-specific polymerase chain reaction (ASPCR) is a cost-saving method that scores SNPs by difference of the PCR efficiency of allele-specific primers. However, ASPCR for SNP typing is notoriously confounded for its locus-specific unpredictability and the laborious gel electrophoresis. In the current study, we investigated the real-time kinetics of ASPCR and found that a simple touchdown thermocycling protocol improved its specificity significantly. Combined with real-time PCR, we developed a homogeneous genotyping method and scored more than 1000 genotypes, including all transition and transversion SNPs. A clear genotyping result was identified and validated the robustness of the method. Optimization of reactions and intrinsic modification of allele-specific primers, a laborious process but one that is repeatedly reported to be inevitable for successful ASPCR, was proved to be unnecessary with our method. Accuracy was confirmed with mass spectrometry. These characters enabled real-time ASPCR with the touchdown thermocycling protocol being very competitive among various SNP typing methods for large-scale genetic studies.

Lymphomatoid papulosis histopathologically simulating angiocentric and cytotoxic T-cell lymphoma: a case report.

We report a patient presenting with a 20-year history of recurrent papulonecrotic lesions in which skin biopsy shows extensive vascular destruction. Atypical lymphoid cells surrounding the dermal vessels had a CD3+, CD4-, CD5-, CD8+, CD20-, CD30+, CD56+, TIA-1+, and granzyme B immunophenotype implicating a natural killer/T origin. In situ hybridization was negative for Epstein-Barr virus transcripts. Analysis of T-cell receptor-gamma gene of 2 separate biopsy specimens detected an identical clone. The patient was treated with low-dose methotrexate and achieved complete resolution in a month. According to the clinical course, immunophenotype, clonality analysis and the excellent response to methotrexate, we conclude that this is an unusual case of lymphomatoid papulosis. We believe that this unusual presentation needs to be distinguished from other aggressive lymphomas, including the natural killer/T-cell and cytotoxic T-cell subsets.

Annular pustules in Kawasaki disease: a further case indicating the association with psoriasis?

We report the case of a 4-year-old girl with Kawasaki disease (KD), or mucocutaneous lymph node syndrome, who presented with an annular pustular eruption. Targetlike erythematous and scaly patches were observed after resolution of the pustules. A biopsy of the skin was performed, and results showed spongy pustules not associated with the intraepidermal eccrine duct. Generalized pustular eruption, including pustular psoriatic lesions, has been described in KD. However, to our knowledge, this is the first report of annular pustular eruption mimicking annular pustular psoriasis in KD.

Efficacy and safety of topically applied tacrolimus ointment in patients with moderate to severe atopic dermatitis.

BACKGROUND: Tacrolimus is a T-cell-selective cytokine inhibitor. The topical formulation was specifically developed for treating inflammatory skin diseases, including atopic dermatitis (AD). The purpose of this study was to evaluate the efficacy and safety of topically applied tacrolimus ointment (Protopic) in treating Taiwanese patients with moderate to severe AD. METHODS: This was an open-labeled, non-comparative, single-center study. Enrolled patients applied Protopic twice daily for up to 4 weeks. Efficacy was evaluated on the basis of the physician's global evaluation of clinical response (PG). Other evaluations included the Eczema Area and Severity Index and the percentage of body surface area; the total score for clinical signs was used for evaluating the efficacy. A safety assessment was based on adverse events reported by patients or observed by the physician. Blood was sampled for the evaluation of tacrolimus levels. RESULTS: In total, 30 patients (16 pediatric and 14 adult patients) with moderate to severe AD were enrolled in this study. At the end of treatment, the PG was 92.3% for the pediatric group and 100% for the adult group. Other efficacy evaluations also showed significant improvement. Two patients had detectable blood tacrolimus concentrations. Skin itching and burning on the application site was a common but temporary side effect. No significant adverse events related to Protopic were observed. CONCLUSION: The results of the study suggest that Protopic ointment may be a safe and effective therapy for treating patients at least 2 years of age with moderate to severe AD.