Evaluation of an amplicon-based custom gene panel for the diagnosis of hereditary tumors.

Genetic testing based on next-generation sequencing (NGS) analysis has recently been used to diagnose hereditary diseases. In this study, we explored the usefulness of our custom amplicon panel that targeted 23 genes related to hereditary tumors given in the American College of Medical Genetics and Genomics recommendations. We applied our custom NGS panel to samples from 12 patients previously diagnosed by Sanger sequencing as having the diseases or diagnosed clinically by meeting the diagnostic criteria in this study. Our gene panel not only successfully identified all variants detected by Sanger sequencing but also identified previously unrecognized variants that resulted in confirmation of the disease, or even in the revision of the diagnosis. For instance, a patient identified with an SDHD gene mutation actually had von Hippel-Lindau (VHL) syndrome, as determined by the presence of a pathogenic VHL gene variant. We also identified false-positive results that were generated by amplification of genome regions that are not intended to be investigated. In conclusion, NGS-based amplicon sequencing is a highly effective method to detect germline variants, as long as they are also carefully reviewed by manual inspection.

Measurement of small fibre pain threshold values for the early detection of diabetic polyneuropathy.

AIM: To investigate whether Aδ and C fibre pain threshold values, measured using intra-epidermal electrical stimulation (IES), in people with and without Type 2 diabetes are useful in evaluating diabetic polyneuropathy (DPN) severity. METHODS: Aδ and C fibre pain threshold values were measured in Japanese people with (n = 120) and without (n = 76) Type 2 diabetes by IES. Nerve conduction studies and other tests were performed to evaluate diabetic complications. RESULTS: Aδ and C fibre pain threshold values were high in people with diabetes compared with control subjects (Aδ fibre: 0.050 vs. 0.030 mA, P < 0.01; C fibre: 0.180 vs. 0.070 mA, P < 0.01). Participants with diabetes and neuropathy had significantly higher Aδ and C fibre pain threshold values than participants without neuropathy (Aδ fibres 0.063 vs. 0.039 mA, P < 0.01; C fibres 0.202 vs. 0.098 mA, P < 0.05). C fibre pain threshold values were significantly higher in participants with diabetes and diabetic microvascular complications than in participants without complications. Threshold values increased with complication progression. When DPN was diagnosed according to the Diabetic Neuropathy Study Group in Japan criteria, the cut-off for the C fibre pain threshold values was 0.125 mA (area under the curve 0.758, sensitivity 81.5%, specificity 61.5%). The IES test took less time (P < 0.01) and was less invasive (P < 0.01) than the nerve conduction studies. CONCLUSIONS: Intra-epidermal electrical stimulation is a non-invasive and easy measurement of small fibre pain threshold values. It may be clinically useful for C fibre measurement to diagnose early DPN as defined by the Diabetic Neuropathy Study Group in Japan criteria.

Structural, enzymatic and molecular studies in a series of nonbullous congenital ichthyosiform erythroderma patients.

Causative gene defects have not been demonstrated in the majority of nonbullous congenital ichthyosiform erythroderma (NBCIE) cases. The purpose of this study was to further elucidate the pathogenesis of NBCIE. Immunohistochemical and ultrastructural observations, transglutaminase activity assays and sequencing of TGM1 were performed in five patients from four NBCIE families. Transglutaminase 1 (TGase 1), involucrin and loricrin expression and in situ transglutaminase activity were present in all of the cases. Ultrastructurally, two cases out of five showed incomplete thickening of the cornified cell envelope (CCE) during keratinization and the other three exhibited abnormal lipid droplets in the cornified cells and malformed lamellar granules. No TGM1 mutation was found in any of the four families by direct sequence analysis. NBCIE cases with normal TGase 1 seemed to have two distinct patterns of abnormality, one with abnormal lipid droplets and malformed lamellar granules and the other with defective CCE formation.

Insulin down-regulates resistin mRNA through the synthesis of protein(s) that could accelerate the degradation of resistin mRNA in 3T3-L1 adipocytes.

AIMS/HYPOTHESIS: Resistin is a peptide secreted by adipocytes and recognized as a hormone that could link obesity to insulin resistance. This study was designed to examine the effect and mechanism(s) of insulin on resistin expression in 3T3-L1 adipocytes. METHODS: Differentiated 3T3-L1 adipocytes were stimulated with insulin and resistin mRNA expression was examined by Northern blot analysis. In some experiments, the insulin signal was blocked by several chemical inhibitors or overexpression of a dominant negative form (Deltap85) of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase). RESULTS: Insulin treatment caused a reduction of resistin mRNA in time-dependent and dose-dependent manners in 3T3-L1 adipocytes. Pre-treatment with PD98059, an inhibitor of extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, or SB203580, an inhibitor of p38 mitogen-activated protein-kinase (p38 MAP-kinase) pathway, did not influence insulin-induced reduction of resistin mRNA. Inhibition of PI 3-kinase by LY294002 or Deltap85 also failed to block insulin-induced reduction of resistin mRNA. Cycloheximide, a protein synthesis inhibitor, completely blocked insulin-induced reduction of resistin mRNA. Actinomycin D, a RNA synthesis inhibitor, also blocked insulin-induced reduction of resistin mRNA, and the decreasing rate of resistin mRNA in cells treated with insulin alone was faster than that with actinomycin D. CONCLUSION/INTERPRETATION: Insulin downregulates resistin mRNA via PI 3-kinase, ERK or p38 MAP-kinase independent pathways in 3T3-L1 adipocytes. The downregulation mechanism of resistin mRNA by insulin would be an indirect event through the synthesis of novel protein(s) that could accelerate the degradation of resistin mRNA.

Heterozygous knockout of the IRS-1 gene in mice enhances obesity-linked insulin resistance: a possible model for the development of type 2 diabetes.

Insulin receptor substrate 1 (IRS-1) gene polymorphisms have been identified in type 2 diabetic patients; however, it is unclear how such polymorphisms contribute to the development of diabetes. Here we introduced obesity in heterozygous IRS-1 knockout (IRS-1(+/-)) mice by gold-thioglucose (GTG) injection and studied the impact of reduced IRS-1 expression on obesity-linked insulin resistance. GTG injection resulted in approximately 30% weight gain in IRS-1(+/-) and wild type (WT) mice, compared with saline-injected controls. There was no difference in insulin sensitivity between lean IRS-1(+/-) and lean WT. Elevated fasting insulin levels but no change in fasting glucose were noted in obese IRS-1(+/-) and WT compared with the respective lean controls. Importantly, fasting insulin in obese IRS-1(+/-) was 1.5-fold higher (P<0.05) than in obese WT, and an insulin tolerance test showed a profound insulin resistance in obese IRS-1(+/-) compared with obese WT. The islets of obese IRS-1(+/-) were 1.4-fold larger than those of obese WT. The expression of insulin receptor and IRS-1 and IRS-2 was decreased in obese IRS-1(+/-), which could in part explain the profound insulin resistance in these mice. Our results suggest that IRS-1 is the suspected gene for type 2 diabetes and its polymorphisms could worsen insulin resistance in the presence of other additional factors, such as obesity.

Identification of a cis-acting element and a novel trans-acting factor of the human insulin receptor gene in HepG2 and rat liver cells.

The liver is a major target organ of insulin and is important for glucose homeostasis. We analyzed the tissue specific regulation of the insulin receptor gene in the liver by studying the cis-acting element and trans-acting factor of the human insulin receptor gene in human hepatoma cell line, HepG2 cells. In the chloramphenicol acetyl transferase (CAT) assay with chimeric plasmids containing various deletions and insertions of the human insulin receptor promoter/CAT gene, a HepG2 cell specific cis-acting element was identified between nt -592 to -577 of the promoter. In electrophoretic mobility shift assay and UV cross-link analysis, a 35-kDa nuclear protein that bound to 5'-TCCCTCCC-3' (nt -588 to -581) sequence was identified in HepG2 cells as well as in rat hepatocytes. This nuclear protein, designated as hepatocyte-specific transcription factor of the insulin receptor gene (HTFIR), might play an important role in tissue-specific expression of the insulin receptor gene in the liver.

Palladium-catalyzed cross-coupling of silanols, silanediols, and silanetriols promoted by silver(I) oxide

Palladium-catalyzed cross-coupling of aryl- or alkenylsilanols, silanediols, and silanetriols with a variety of iodoarenes by the catalysis of palladium(0) and in the presence of silver(I) oxide furnished the coupling products in good to excellent yields. The reactions of silanediols or silanetriols under similar conditions proceeded much faster than those of silanols to afford the corresponding coupling products in excellent yields within shorter reaction periods (5-12 h). The measurement of the X-ray diffraction (XRD) pattern of the silver residue after the reaction revealed that silver(I) oxide was converted to silver(I) iodide.

Non-Sonogashira-type palladium-catalyzed coupling reactions of terminal alkynes assisted by silver(I) oxide or tetrabutylammonium fluoride.

Palladium-catalyzed reaction of aryl and alkenyl halides with terminal alkynes in the presence of silver(I) oxide as an activator furnishes various arylated or alkenylated alkynes in good to excellent yields. The similar coupling reaction is also found to proceed using tetrabutylammonium fluoride (TBAF) or tetrabutylammonium hydroxide (TBAOH) as an activator.

Bradykinin enhances insulin receptor tyrosine kinase in 32D cells reconstituted with bradykinin and insulin signaling pathways.

We have previously shown that bradykinin potentiated insulin-induced glucose uptake through GLUT4 translocation in canine adipocytes and skeletal muscles. The aim of this study was to determine the molecular mechanism of bradykinin enhancement of the insulin signal. For this purpose, 32D cells, which express a limited number of insulin receptors and lack endogenous bradykinin B2 receptor (BK2R) or insulin receptor substrate (IRS)-1 were transfected with BK2R cDNA and/or insulin receptor cDNA and/or IRS-1 cDNA, and analyzed. In 32D cells that expressed BK2R and insulin receptor (32D-BKR/IR), bradykinin alone had no effect on the phosphorylation of the insulin receptor, but it enhanced insulin-stimulated tyrosine phosphorylation of the insulin receptor. In 32D cells that expressed BK2R, insulin receptor and IRS-1 (32D-BKR/IR/IRS1), bradykinin also enhanced insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS-1. An increase in insulin-stimulated phosphorylation of IRS-1 by treatment with bradykinin in 32D-BKR/IR/IRS1 cell was associated with increased binding of 85 kD subunit of phosphatidylinositol 3 (PI 3)-kinase and increased IRS-1 associated PI 3-kinase activity. These effects of bradykinin were not observed in 32D cells which lack the expression of BK2R (32D-IR/IRS1) or insulin receptor (32D-BKR/IRS1). Furthermore, tyrosine phosphatase activity against insulin receptor beta-subunit in plasma membrane fraction of 32D-BKR/IR cells was significantly reduced by bradykinin, suggesting that the effect of bradykinin was in part mediated by inhibition of protein tyrosine phosphatase(s). Our results clearly demonstrated that bradykinin enhanced insulin-stimulated tyrosine kinase activity of the insulin receptor and downstream insulin signal cascade through the BK2R mediated signal pathway.

[Randomized controlled trial for smoking cessation among city office employees].

To evaluate the effect of a smoking cessation program by health professionals, a randomized intervention study was carried out in the Omihachiman city office in 1993. Participants (n = 53), volunteers from current smokers in the city office, were randomly divided into intervention and control groups. The intervention group received intensive education for five months (i.e., the effect of smoking on health, the beneficial aspects of quitting smoking, how to stop smoking and how to deal with the withdrawal symptoms). Group lectures (two times) and individual counseling (three times) were used for health education. After five months, the control group was also given the same advice on smoking cessation. Comparison of smoking cessation rates between the two groups was performed at the end of the intervention period. Follow-up of all participants occurred at six and 12 months post intervention. After the five months of intervention, smoking cessation rate in the intervention group (19.2%) tended to be higher than that in the control group (7.4%), but was not significant (chi 2 = 1.62). Over all smoking cessation rates of all participants (n = 53) at the end of the 10 month intervention was 32.1% and at six months and 1 year after the end of the 10 month intervention were 24.5% and 13.2%, respectively. Comparison of participants who successfully stopped smoking and those who did not, it was revealed that younger age, lower expired air CO concentration (p < 0.01), and attitude for smoking cessation at the beginning were significantly related to smoking cessation. In our study, after five months, smoking cessation rate in the intervention group was about two times that of the control group, although the effectiveness of our smoking cessation program could not be validated due to small sample size. Taking into account the rate of smoking cessation after one year, We believe that programs by health professionals are effective for smoking cessation.

The effect of high-salt diet intake on muscular exercise ability in young Japanese women.

This study was designed to test whether high-salt diet intake has some acute impaired effect to the muscular exercise ability due to the calcium deficit in muscle cell via the accelerated sodium-calcium exchanger. Six healthy young Japanese women (aged: 22.3 +/- 1.9 yr) performed two types of muscle strength tests and ramp mode cycle ergometer exercise until exhaustion after normal- (NaCl is approximately 5.6 g) and high-salt (21.0 g) controlled diet intake on two separate days in random order. The urinary sodium excretion sampled during 12 hours on the high-salt diet day was significantly higher compared to that of normal-salt diet day (3301 +/- 992 vs 1595 +/- 540 mg; P < 0.05), while there was no substantial difference between the urinary calcium excretion in high- and normal-salt diet days (58.6 +/- 19.7 vs 55.0 +/- 17.2 mg; ns). There were no significant differences in back strength, repeated maximal hand grip exercise ability, and VO2max and duration time during ramp exercise between high- and normal-salt diet conditions. It was concluded that high-salt diet intake even exceeding 20 g per day had substantially no acute effect on muscular exercise ability in young Japanese women.

Self-involuting atrophoderma of the lateral-upper arm. A new benign variant of morphea?

BACKGROUND: Atrophoderma of Pasini-Pierini and morphea are considered to be distinct clinical entities. However, some authors say that they are closely related diseases. We encountered 5 unique cases that did not fit the criteria for both diseases. OBJECTIVE AND METHODS: We report 5 cases with solitary atrophic lesions on the lateral-upper arm. They appeared as white to erythematous, non-indurated and slightly depressed lesions with a smooth surface, and the patients had no history of trauma or injection. RESULTS: Histological examinations showed slight to moderate lower dermal fibrosis without specific changes in adipose tissue. The lesions disappeared spontaneously within a year. CONCLUSION: Our cases may be a benign variant form of morphea. We propose the term 'self-involuting atrophoderma of the lateral-upper arm (SALA)' for these clinical features.