A novel stop-gain NF1 variant in neurofibromatosis type 1 and bilateral optic atrophy without optic gliomas.

BACKGROUND: Neurofibromatosis type 1 (NF1) is a multisystem disorder that primarily affects the skin and peripheral nervous system and is caused by chromosomal abnormalities and mostly truncating variants in the NF1 gene. Ocular complications such as Lisch nodules and optic pathway gliomas (OPGs) can occur in NF1 patients. Herein, we report a novel NF1 variant in an NF1 patient with bilateral optic atrophy. METHODS: Ophthalmological examinations and genetic analyses were performed using targeted next-generation sequencing (NGS). RESULTS: A 14-year-old girl diagnosed with NF1 visited our hospital with decreased visual acuity (VA). The patient had no family history of NF1 or visual impairment. Brain and orbital magnetic resonance imaging revealed no remarkable findings. Ophthalmoscopy revealed temporal pallor of the optic discs, which was confirmed by optical coherence tomography findings of significant thinning of the circumpapillary retinal nerve fiber layer in both eyes. At 23 years of age, the decimal-corrected VA had deteriorated to 0.2 in the right eye and 0.1 in the left eye. Additionally, the targeted NGS panel revealed a novel heterozygous stop-gain variant (p.Tyr628Ter) in the NF1 gene; however, no pathogenic variants in OPA1 or the mitochondrial DNA were identified. CONCLUSIONS: A patient with NF1 without OPGs developed bilateral optic atrophy and carried a novel de novo stop-gain variant of NF1. Although the relationship between NF1 variants and bilateral optic atrophy remains unclear, further investigations are required.

Association of pulse pressure with all-cause mortality in older Japanese patients with type 2 diabetes mellitus: A observational cohort study.

AIM: Although systolic and diastolic blood pressures as well as blood glucose are monitored when nurses care for patients with type 2 diabetes, the same is not true for pulse pressure. We aimed to determine the association between pulse pressure and all-cause mortality. METHODS: We conducted a longitudinal study of outpatients with type 2 diabetes aged 65 years and older at diabetes-specialized hospitals in Japan from September 2004 to December 2016. Descriptive data, blood pressure measurements, blood analysis data, and information on life and death were obtained from medical records. Cox proportional hazards models were used to estimate the relative risks with 95% confidence intervals for all-cause mortality. RESULTS: We analyzed 357 of the 383 recruited patients (mean age, 74.9 years; 175 men and 182 women; average follow-up, 7.7 years), and 50 patients died. After adjusting for covariates, the relative risks for pulse pressures of 55 to <65, 65 to <75, and ≥75 mmHg (reference: <55 mmHg) were 1.77 (95% confidence interval: [0.59, 5.28]), 2.66 (95% confidence interval: [0.93, 7.56]), and 3.23 (95% confidence interval: [1.16, 8.99]), respectively. The relative risk for the 65 mmHg or higher group (reference: <65 mmHg) was 2.08 (95% confidence interval: [1.11, 3.92]). Neither systolic blood pressure nor diastolic blood pressure alone were significantly associated with mortality. CONCLUSIONS: In older patients with type 2 diabetes, a wide pulse pressure was associated with a higher risk of all-cause mortality. Nurses caring for older people with diabetes should also monitor pulse pressure.

Characterization of the hepatic cellular uptake of α(1) -acid glycoprotein (AGP), part 1: a peptide moiety of human AGP is recognized by the hemoglobin ß-chain on mouse liver parenchymal cells.

Human α(1) -acid glycoprotein (AGP), a serum glycoprotein, is known to have anti-inflammatory activity. We recently reported that AGP was mainly incorporated into the liver in mice via a receptor-mediated pathway, although the mechanism for this was largely unknown. The objective of this study was to identify the specific cellular surface protein that recognizes the peptide moiety of AGP. Pharmacokinetic studies of (111) In-AGP and (111) In -recombinant glycan-deficient AGP (rAGP) in mice demonstrated that both AGPs are mainly distributed to the liver and kidney, but hepatic and renal uptake clearance of rAGP was higher than that for AGP. Hepatic uptake of rAGP was inhibited in the presence of 100-fold excess of unlabeled AGP, indicating that the hepatic uptake of rAGP shared a common route with that of AGP and that it recognized the peptide moiety of AGPs. In ligand blotting analyses using crude cellular membrane fraction of mice liver, a band corresponding to a 16 kDa protein was observed to bind to both AGPs. Interestingly, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry MALDI-TOF-MS and western blotting analyses indicated that this 16 kDa protein is the hemoglobin ß-chain (HBB). It, therefore, appears that HBB is associated with the hepatic uptake of AGP via a direct interaction with its peptide moiety.

Structural insights into differences in drug-binding selectivity between two forms of human alpha1-acid glycoprotein genetic variants, the A and F1*S forms.

Human α(1)-acid glycoprotein (hAGP) in serum functions as a carrier of basic drugs. In most individuals, hAGP exists as a mixture of two genetic variants, the F1*S and A variants, which bind drugs with different selectivities. We prepared a mutant of the A variant, C149R, and showed that its drug-binding properties were indistinguishable from those of the wild type. In this study, we determined the crystal structures of this mutant hAGP alone and complexed with disopyramide (DSP), amitriptyline (AMT), and the nonspecific drug chlorpromazine (CPZ). The crystal structures revealed that the drug-binding pocket on the A variant is located within an eight-stranded ß-barrel, similar to that found in the F1*S variant and other lipocalin family proteins. However, the binding region of the A variant is narrower than that of the F1*S variant. In the crystal structures of complexes with DSP and AMT, the two aromatic rings of each drug interact with Phe-49 and Phe-112 at the bottom of the binding pocket. Although the structure of CPZ is similar to those of DSP and AMT, its fused aromatic ring system, which is extended in length by the addition of a chlorine atom, appears to dictate an alternative mode of binding, which explains its nonselective binding to the F1*S and A variant hAGPs. Modeling experiments based on the co-crystal structures suggest that, in complexes of DSP, AMT, or CPZ with the F1*S variant, Phe-114 sterically hinders interactions with DSP and AMT, but not CPZ.

Construction of an expression system for human alpha(1)-acid glycoprotein in E. coli: The roles of oligosaccharide moieties in structural and functional properties.

Unglycosylated recombinant human alpha(1)-acid glycoprotein (hAGP) variants (rF1(*)S and rA) were prepared in an E. coli expression system using the Origami B strain and pET-3c vector. Thioredoxin was co-expressed to promote the appropriate folding of hAGP. SDS-PAGE under reducing conditions showed that rF1(*)S and rA migrate as single bands after purification. However, several bands derived from rA were observed under non-reducing conditions because of the high reactivity of a free cystein residue (C149). We therefore prepared a mutant of A variant (C149R-A), and confirmed that this mutant maintained homogeneity. Circular dichroism and intrinsic tryptophan fluorescence spectroscopic analyses indicated that rF1(*)S and C149R-A have almost the same conformational structures as F1(*)S and A purified from serum. Ligand binding experiments using propranolol as a F1(*)S ligand and disopyramide as an A specific ligand indicated that the capacity of rF1(*)S and C149R-A is equivalent to those ligands as well as F1(*)S and A from serum. These results suggest that the oligosaccharide moieties of hAGP have negligible effects on the structural and ligand binding properties of hAGP. Thus, rF1(*)S and C149R-A promise to be useful in studies on the drug binding sites of hAGP.

Correlation between voriconazole trough plasma concentration and hepatotoxicity in patients with different CYP2C19 genotypes.

Voriconazole metabolism is mostly mediated via the cytochrome P450 (CYP) 2C19 isozyme. The non-wild (mutant) type of CYP2C19 is generally found in 60-70% of Asian populations. Because the voriconazole trough plasma concentration has been reported to correlate with hepatotoxicity, this study investigated the effect of CYP2C19 polymorphism on the relationship between voriconazole trough concentrations and liver function abnormalities in 29 Japanese patients with fungal infections (CYP2C19 wild-type, n=10; non-wild-type, n=19). Hepatotoxicity, defined as liver enzyme abnormality according to the National Cancer Institute criteria, was observed in 10 (34.5%) of 29 patients with a trough concentration > or = 3.9 mg/L. Logistic regression analysis suggested that the therapeutic range for the voriconazole trough concentration should be 2-4 mg/L. Non-linear pharmacokinetic analysis suggested that voriconazole therapy should be initiated with a dose of 7.2-8.9 mg/kg/day for CYP2C19 wild-type and 4.4-6.5mg/kg/day for the non-wild-type in Japanese patients. These recommended initial dosages and subsequent dose adjustment for the target concentration range by therapeutic drug monitoring should avoid adverse events and thus enable continued effective voriconazole therapy for Japanese patients with mycoses.

A site-directed mutagenesis study of drug-binding selectivity in genetic variants of human alpha(1)-acid glycoprotein.

Human alpha(1)-acid glycoprotein (AGP), a major carrier of many basic drugs in circulation, consists of at least two genetic variants, namely A and F1*S variant. Interestingly, the variants of AGP have different drug-binding properties. The purpose of this study was to identify the amino acid residues that are responsible for the selectivity of drug binding to genetic variants of AGP using site-directed mutagenesis. First, we screened amino acid residues in the region proximal to position 100 that are involved in binding of warfarin and dipyridamole, which are F1*S-specific ligands, and of propafenone, which is an A-specific ligand, using ultrafiltration. In the F1*S variant, His97, His100, and Trp122 were involved in either warfarin- or dipyridamole-binding, while Glu92, His100, and Trp122 participated in the binding of propafenone in the A variant. Exchange of the residue at position 92 between AGP variants reversed the relative strength of propafenone binding to the two variants, but had a markedly different effect on binding of warfarin and dipyridamole. These findings indicate that the amino acid residue at position 92 plays a significant role in drug-binding selectivity in AGP variants, especially for drugs that preferentially bind to the A variant.

Involvement of disulfide bonds and histidine 172 in a unique beta-sheet to alpha-helix transition of alpha 1-acid glycoprotein at the biomembrane interface.

Human alpha(1)-acid glycoprotein (AGP), which is comprised of 183 amino acid residues and 5 carbohydrate chains, is a major plasma protein that binds to basic and neutral drugs as well as to steroid hormones. It has a beta-sheet-rich structure in aqueous solution. Our previous findings suggest that AGP forms an alpha-helix structure through an interaction with biomembranes. We report herein on a study of the mechanism of alpha-helix formation in AGP using various modified AGPs. The disulfide reduced AGP (R-AGP) was extensively unfolded, whereas asialylated AGP (A-AGP) maintained the native structure. Intriguingly, reduced and asialylated AGP (RA-AGP) increased the alpha-helix content as observed in the presence of biomembrane models, and showed a significant decrease in ligand binding capacity. This suggests that AGP has an innate tendency to form an alpha-helix structure, and disulfide bonds are a key factor in the conformational transition between the beta-sheet and alpha-helix structures. However, RA-AGP with all histidine residues chemically modified (HRA-AGP) was found to lose the intrinsic ability to form an alpha-helix structure. Furthermore, disulfide reduction of the H172A mutant expressed in Pichia pastoris also caused a similar loss of folding ability. The present results indicate that disulfide bonds and the C-terminal region, including H172 of AGP, play important roles in alpha-helix formation in the interaction of the protein with biomembranes.

Construction of expression system for human alpha 1-acid glycoprotein in Pichia pastoris and evaluation of its drug-binding properties.

Human alpha1-acid glycoprotein (hAGP) is a plasma glycoprotein that functions as a major carrier of basic ligands. This is the first report of the recombinant hAGP (rhAGP). In this study, rhAGP was expressed in the methylotropic yeast Pichia pastoris (GS115) using the expression vector, pPIC9, and then purified by anionic exchange, hydrophobic interaction, and gel filtration chromatography. The molecular weight of rhAGP was much lower than that of hAGP, because of the difference in glycan chain content. Results of glycopeptidase F digestion suggest that the peptide moiety of rhAGP was the same as that of hAGP. The results of circular dichroism spectra measurement indicated that rhAGP predominantly formed a beta-sheet-rich structure that was the same as that of hAGP and typical of the lipocalin family. From the experiments using AGP-binding drugs (chlorpromazine, warfarin, and progesterone) and quinaldine red as a probe for the binding site, it was indicated that rhAGP also had the same ligand-binding capacity and binding site structure as hAGP. These findings strongly suggest that this recombinant hAGP (rhAGP) is very useful for the exploration of the ligand-binding site and biological function of hAGP.