Diagnostic strategy for inherited hypomagnesemia

Background

Hereditary hypomagnesemia is difficult to diagnose accurately because of its rarity and the variety of causative genes. We established a flowchart for identifying responsible genes for hypomagnesemia, and we confirmed its diagnostic efficacy in patients with suspected inherited hypomagnesemia.

Methods

We established a flowchart and applied it to five index cases with suspected inherited hypomagnesemia. Direct sequence analysis was used to detect the causative gene variants in four cases, and targeted sequencing analysis using next-generation sequencing (NGS) of all causative genes for hypomagnesemia was used in one.

Results

Expected pathogenic variants were detected in the HNF1B, TRPM6, CLDN16, CASR, or SLC12A3 gene in all five cases. The results of all genetic analyses were consistent with the clinical diagnostic results using the flowchart.

Conclusions

Accurate genetic diagnosis is crucial for estimating the prognosis, detecting complications in organs other than the kidneys, and for directing genetic counseling. The developed flowchart for identifying responsible genes for hypomagnesemia was useful for diagnosing inherited hypomagnesemia. In addition, NGS analysis will help to resolve clinical difficulties in making an accurate diagnosis and thus improve the diagnostic strategy for inherited hypomagnesemia.

     

Correlation between parametric imaging using contrast ultrasound and the histological differentiation of hepatocellular carcinoma.

Aim: To determine whether parametric imaging correlates with the degree of histological differentiation of hepatocellular carcinoma (HCC). Methods: The samples comprised 49 nodules diagnosed histologically as HCC: 19 well differentiated (w-HCC), 22 moderately differentiated (m-HCC), and eight poorly differentiated (p-HCC). The ultrasound (US) equipment used was SSA-770 A (Toshiba Medical Systems, Otawara, Japan) and the contrast agent was SonoVue (Bracco, Milan, Italy). After 1.5 mL of SonoVue was injected intravenously and staining of the tumors and parenchyma was confirmed, microbubbles in the scanned volume were eliminated using high mechanical index (MI) scanning frames. The "arrival time (T(A)) images," reflecting beta-values, were displayed with color codes at the phase after reperfusion. Images at the phase when the staining reached a plateau (90-180 s) were used as "A images," reflecting A values. These images were compared between each histological grade of differentiation. Results: Analysis of T(A) images indicated that beta-values in m-HCC were higher than those in the adjacent non-tumor parenchyma in all 22 samples and also were significantly higher than in the other HCCs (P < 0.001 for w-HCC; P < 0.05 for p-HCC). Furthermore, beta-values in p-HCC samples had significantly larger variations in terms of time and space than in the other HCCs (P < 0.001 for w-HCC; P < 0.01 for m-HCC). Analysis of A images indicated that the A value for w-HCC was significantly higher than those for either m-HCC or p-HCC (P < 0.001). Conclusion: Both T(A) and A images were useful for diagnosing the histological differentiation of HCC.     

Stability for long-term storage and reproducibility of positivity in the panel test slide prepared with the polyacrylamide-based artificial sputum

OBJECTIVE: A novel artificial sputum has been developed using polyacrylamide, cultured THP-1 cell and BCG-Pasteur. Smears prepared with this artificial sputum are similar to actual sputum and has feasibility to set any positivity grades. Long-term storage and reproducibility of the positivity was examined to support further availability. METHOD: The artificial sputa were stored for up to 9 months at room temperature, 4 degrees C and -20 degrees C. Then, smears were prepared and their macroscopic and microscopic appearance were examined compared with smears from freshly prepared artificial sputum. Furthermore, smears with different positivities (+/-, 1 +, 2 + and 3 +) were prepared and examined by several trained technicians, and the reproducibility of the original sputum positivity was determined. RESULTS: Macroscopic and microscopic appearance of smears prepared from long-term stored artificial sputum showed little changes compared with smears of freshly prepared artificial sputum. The positivity of these smears fell in their original grade. A total of 91 smears were prepared from artificial sputum with different positivity and examined by trained technicians. Although 3 out of 36 +/- smears were determined as negative, all of the remaining smears were evaluated correctly. DISCUSSION: This study confirmed that the artificial sputum and the smears have long-term storage stability and reproducibility in the positivity. These results suggest that the artificial sputum can be widely used to perform external quality assessment in many countries, including high prevalence countries.     

Preventive effect of N-acetylcysteine on contrast-induced nephropathy following coronary angiography and angioplasty

Contrast-induced nephropathy (CIN) is one of the serious side effects of contrast media. A few studies have suggested that N-acetylcysteine (NAC) is effective to prevent CIN, but the efficacy remains unclear in Japanese. Therefore, we retrospectively studied the preventive effect of NAC on CIN in Sakakibara Heart Institute. Patients who had been administered NAC for the purpose of preventing CIN before coronary intervention between February 2005 and November 2006 were included in the NAC group. In addition, age- and rate of diabetes mellitus-matched controls were randomly extracted. We retrieved and analyzed patient data including demographics, NAC dosage, and serum creatinine concentrations (Scr). NAC group (n=16) showed significantly higher baseline Scr (p<0.01) and a tendency toward a lower dose of contrast media (p=0.068) compared with controls (n=48). Since the occurrence of CIN was low, there was no significant difference in the proportion of CIN between the groups (NAC: 6%, controls: 4%). NAC group trended toward a decrease in Scr after the use of contrast media, while controls increased (-0.04+/-0.25 versus +0.03+/-0.36 mg/dl, p=0.096). The multivariate analysis showed that the dosage of NAC is inversely correlated with Scr independent of baseline Scr and dosage of contrast media. Despite higher baseline Scr (i.e., high-risk with CIN) in the NAC group, the real Scr value reflected a lower trend on average. In addition, this finding suggests that a larger dose of NAC results in a lower Scr value, we consider that the NAC dosage more likely prevented CIN.     

Characterization of meFucoidan as a selective inhibitor for secretory phospholipase A2-IIA and the phosphorylation of meFucoidan-binding proteins by A-kinase in vitro

The direct interaction of Mekabu fucoidan (meFucoidan) with four functional basic proteins (sPLA2-IIA, bFGF, histone H2B and HBV core protein) and three synthetic FGF-BP peptides (sp5, GE13 and RS6) was characterized in vitro. It was found that (i) meFucoidan inhibited dose-dependently the activity of sPLA2-IIA, but not pPLA2, through its direct binding to the enzyme; (ii) sPLA2-IIA activity was sensitive to meFucoidan rather than heparin, but significantly stimulated by sulfatide; (iii) the A-kinase-mediated phosphorylation of these basic proteins, except sPLA2-IIA, and synthetic peptides, containing potent phosphorylation sites for A-kinase, was inhibited dose-dependently by meFucoidan; and (iv) two consensus meFucoidan-binding motifs (B-B-B-B-X and B-X-B-B-X; B, basic amino acid) in these basic proteins and synthetic peptides could be overlapping to the potent phosphorylation site (B-B-X-S/T) for the kinase in vitro. These results presented here suggest that meFucoidan functions as a selective inhibitor for sPLA2-IIA and the A-kinase-mediated phosphorylation of cellular meFucoidan-binding functional basic proteins in vitro.     

The crucial protective role of glutathione against tienilic acid hepatotoxicity in rats

To investigate the hepatotoxic potential of tienilic acid in vivo, we administered a single oral dose of tienilic acid to Sprague-Dawley rats and performed general clinicopathological examinations and hepatic gene expression analysis using Affymetrix microarrays. No change in the serum transaminases was noted at up to 1000 mg/kg, although slight elevation of the serum bile acid and bilirubin, and very mild hepatotoxic changes in morphology were observed. In contrast to the marginal clinicopathological changes, marked upregulation of the genes involved in glutathione biosynthesis [glutathione synthetase and glutamate-cysteine ligase (Gcl)], oxidative stress response [heme oxygenase-1 and NAD(P)H dehydrogenase quinone 1] and phase II drug metabolism (glutathione S-transferase and UDP glycosyltransferase 1A6) were noted after 3 or 6 h post-dosing. The hepatic reduced glutathione level decreased at 3-6 h, and then increased at 24 or 48 h, indicating that the upregulation of NF-E2-related factor 2 (Nrf2)-regulated gene and the late increase in hepatic glutathione are protective responses against the oxidative and/or electrophilic stresses caused by tienilic acid. In a subsequent experiment, tienilic acid in combination with l-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of Gcl caused marked elevation of serum alanine aminotransferase (ALT) with extensive centrilobular hepatocyte necrosis, whereas BSO alone showed no hepatotoxicity. The elevation of ALT by this combination was observed at the same dose levels of tienilic acid as the upregulation of the Nrf2-regulated genes by tienilic acid alone. In conclusion, these results suggest that the impairment of glutathione biosynthesis may play a critical role in the development of tienilic acid hepatotoxicity through extensive oxidative and/or electrophilic stresses.