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Background: Polymerase chain reaction (PCR) analysis using DNA from dried blood spot (DBS) samples on filter paper is a critical technique for spinal muscular atrophy (SMA) newborn screening. However, DNA extraction from DBS is time-consuming, and elimination of PCR inhibitors from DBS is almost impossible., Methods: Exon 7 of the two homologous SMA-related genes, survival motor neuron (SMN) 1 and SMN2, of five SMA patients and five controls were amplified by PCR with a punched-out circle of the DBS paper. Two types of DNA preparation methods were tested; DNA-extraction (extracted DNA was added in a PCR tube) and non-DNA-extraction (a punched-out DBS circle was placed in a PCR tube). As for the DNA polymerases, two different enzymes were compared; TaKaRa Ex Taq™ and KOD FX Neo™. To test the diagnostic quality of PCR products, RFLP (Restriction fragment length polymorphism) analysis with DraI digestion was performed, differentiating SMN1 and SMN2., Results: In PCR using extracted DNA, sufficient amplification was achieved with TaKaRa Ex Taq™ and KOD FX Neo™, and there was no significant difference in amplification efficiency between them. In direct PCR with a punched-out DBS circle, sufficient amplification was achieved when KOD FX Neo™ polymerase was used, while there was no amplification with TaKaRa Ex Taq™. RFLP analysis of the direct PCR products with KOD FX Neo™ clearly separated SMN1 and SMN2 sequences and proved the presence of both of SMN1 and SMN2 in controls, and only SMN2 in SMA patients, suggesting that the direct PCR products with KOD FX Neo™ were of sufficient diagnostic quality for SMA testing., Conclusion: Direct PCR with DNA polymerases like KOD FX NeoTM has potential to be widely used in SMA newborn screening in the near future as it obviates the DNA extraction process from DBS and can precisely amplify the target sequences in spite of the presence of PCR inhibitors.

Objectives: The effect of circulating low-level hepatitis B virus (HBV), defined as one of the states of 'occult HBV infection', on the development of hepatocellular carcinoma (HCC) in HBV surface antigen (HBsAg)-negative patients is controversial. In addition, the prevalence of occult HBV infection strongly depends on the sensitivity of the HBV detection method. We investigated the prevalence of low-level HBV in the serum of HBsAg-negative patients with HCC using a newly developed, sensitive method based on real-time polymerase chain reaction., Methods: Serum was examined for HBV DNA in 132 patients with HBsAg-negative HCC (95 with hepatitis C virus [HCV] infection and 37 without detectable hepatitis virus infection) with the COBAS TaqMan HBV test, of which the 95% hit rate is 35 copies/ml (6.7 IU/ml)., Results: Low-level HBV DNA was detected in 2 of 95 (2.1%) patients with HCV-related HCC and 1 of 37 (2.7%) patients with non-viral HCC., Conclusion: The prevalence of the detection of circulating low-level HBV was low in both HBsAg-negative HCC patients with HCV infection and those without detectable hepatitis virus, even with the use of the most sensitive method for the detection of HBV. Circulating low-level HBV does not appear to play an important role in hepatocarcinogenesis in HBsAg-negative HCC., (Copyright 2007 S. Karger AG, Basel.)

Objective: The molecular basis of neuroleptic malignant syndrome (NMS) is unclear, but clinical studies have noted a genetic predisposition. A recent genetic study suggested an association between NMS and the I A polymorphism in the dopamine D2 receptor (DRD2 ) gene. We further examined the association in a larger number of subjects., Methods: We studied 49 Japanese patients previously diagnosed with NMS, and 123 schizophrenic patients treated with neuroleptics without occurrence of NMS. PCR and RFLP analyses were performed to screen the I A polymorphism., Results: The I A1 allele frequency was 0.408 in NMS patients and 0.415 in patients without NMS. No significant differences in allelic or genotypic frequencies were observed between the two groups., Conclusions: We cannot conclude that the I A polymorphism is associated with development of NMS.

We examined allele frequencies for the common cholesteryl ester transfer protein (CETP) TaqI polymorphisms and the associations of CETP-TaqI polymorphisms with serum lipid and lipoprotein levels taking into account for selected lifestyle factors in a well-characterized random sample of 527 healthy subjects living a rural community in Japan (256 men and 271 women aged 40-69 years). B2 allele frequency was 0.39 in men and 0.41 in women, and its presence was significantly associated with increased levels of HDL cholesterol (HDL-C) in men (P=0.003 for linear trend). A similar tendency in women was observed, although P value for trend did not reach 0.05. There were not significant interactions between TaqIB genotype and smoking and alcohol drinking or daily physical activity in HDL-C. There were no statistically significant differences among TaqIA genotype in lipid and lipoprotein levels. Multiple linear regression analysis showed that B1B2 and B2B2 explained 1.7% and 2.2%, and 0.6% and 1.0% of variation in men and in women in HDL-C, respectively. We conclude that the CETP-TaqIB polymorphism has a quantitative influence, but appears to be stronger one in men, on HDL-C levels even after adjustment for important lifestyle factors (smoking, alcohol drinking, and daily physical activity).

The cause(s) of sarcoidosis is unknown. Mycobacterium spp. are suspected in Europe and Propionibacterium spp. are suspected in Japan. The present international collaboration evaluated the possible etiological links between sarcoidosis and the suspected bacterial species. Formalin-fixed and paraffin-embedded sections of biopsy samples of lymph nodes, one from each of 108 patients with sarcoidosis and 65 patients with tuberculosis, together with 86 control samples, were collected from two institutes in Japan and three institutes in Italy, Germany, and England. Genomes of Propionibacterium acnes, Propionibacterium granulosum, Mycobacterium tuberculosis, Mycobacterium avium subsp. paratuberculosis, and Escherichia coli (as the control) were counted by quantitative real-time PCR. Either P. acnes or P. granulosum was found in all but two of the sarcoid samples. M. avium subsp. paratuberculosis was found in no sarcoid sample. M. tuberculosis was found in 0 to 9% of the sarcoid samples but in 65 to 100% of the tuberculosis samples. In sarcoid lymph nodes, the total numbers of genomes of P. acnes or P. granulosum were far more than those of M. tuberculosis. P. acnes or P. granulosum was found in 0 to 60% of the tuberculosis and control samples, but the total numbers of genomes of P. acnes or P. granulosum in such samples were less than those in sarcoid samples. Propionibacterium spp. are more likely than Mycobacteria spp. to be involved in the etiology of sarcoidosis, not only in Japanese but also in European patients with sarcoidosis.

We have analyzed a mutation responsible for a Hinf I polymorphism of the D1S80 system by Taq dye deoxy terminator cycle sequencing. A G-T transversion at the 58th nucleotide from the 5' end of the forward primer was identified. This mutation has been firmly established by the restriction enzymes. The type G was digested with Hinf I but not with Tsp 509 I. Conversely, the type T was digested with Tsp 509 I but not with Hinj I. We applied this technique to analyze D1S80 system in German and Japanese populations. In the Japanese population, the type T frequencies were much lower than those in the German population, and the type T was almost exclusively observed in the allele 24.