Clinical and mutational spectrum of Charcot-Marie-Tooth disease type 2Z caused by MORC2 variants in Japan.

BACKGROUND AND PURPOSE: The microrchidia family CW-type zinc finger 2 gene (MORC2) was newly identified as a causative gene of Charcot-Marie-Tooth disease (CMT) type 2Z in 2016. We aimed to describe the clinical and mutational spectrum of patients with CMT harboring MORC2 mutations in Japan. METHODS: We analyzed samples from 781 unrelated patients clinically diagnosed with CMT using deoxyribonucleic acid microarray or targeted resequencing by next-generation sequencing, and samples from 434 mutation-negative patients were subjected to whole-exome sequencing. We extracted MORC2 variants from these whole-exome sequencing data and classified them according to American College of Medical Genetics standards and guidelines. RESULTS: We identified MORC2 variants in 13 patients. As the second most common causative gene of CMT type 2 after MFN2, MORC2 variants were detected in 2.7% of patients with CMT type 2. The mean age of onset was 10.3 ± 8.7 years, and the inheritance pattern was mostly sporadic (11/13 patients, 84.6%). The clinical phenotype was typically length-dependent polyneuropathy, and electrophysiological studies revealed sensory-dominant axonal neuropathy. Mental retardation was identified in 4/13 patients (30.8%). p.Arg190Trp, as a mutational hotspot, was observed in eight unrelated families. We also identified two novel probably pathogenic variants, p.Cys345Tyr and p.Ala369Val, and one novel uncertain significance variant, p.Tyr332Cys. CONCLUSIONS: Our study is the largest report of patients harboring MORC2 variants. We revealed a clinical and mutational spectrum of Japanese patients with MORC2 variants. More attention should be paid to cognitive impairment, and the responsible mechanism requires further research for elucidation.

Association of Chr17q25 with cerebral white matter hyperintensities and cognitive impairment: the J-SHIPP study.

BACKGROUND AND PURPOSE: A recent genome-wide association study has successfully identified several genetic variations in the Chr17q25 locus as susceptible genotypes for white matter hyperintensities. We report the first replication study in subjects of non-European origin. We also investigated possible associations with other asymptomatic cerebrovascular diseases and cognitive function. METHODS: Study subjects were 1190 general Japanese persons (66.0 ± 8.9 years old). Asymptomatic cerebrovascular damage, including lacunar infarctions, microbleeds, periventricular hyperintensity and deep and subcortical white matter hyperintensity (DSWMH), was evaluated by brain magnetic resonance imaging. RESULTS: A polymorphism rs3744028 was significantly associated with DSWMH grade (P = 0.015) but not periventricular hyperintensity, lacunar infarction, and microbleeds. Although age, hypertension, insulin resistance, B-type natriuretic peptide, and carotid atherosclerosis were also correlated with DSWMH, association of the genotype was independent of these environmental risk factors. In contrast, the risk allele had a protective effect against reduced cognitive function. CONCLUSION: Susceptibility of the 17q25 locus may be conserved beyond ethnic differences. Genetic variants may have bipolar effects on brain histological and functional changes.

Association between incremental gains in the objective response rate and survival improvement in phase III trials of first-line chemotherapy for extensive disease small-cell lung cancer.

BACKGROUND: The duration of, resources required for and cost of clinical trials could be reduced if a surrogate end point was to be used in place of survival. We assessed the extent to which the objective response rate (ORR) is predictive of mortality, how much difference in the ORR is needed to predict an obvious survival difference and what factors could affect the association between the two parameters during the first-line treatment of extensive disease (ED)-small-cell lung cancer (SCLC). METHODS: We used the ORRs and median survival times (MSTs) from 48 phase III trials of first-line chemotherapy involving 8779 randomised patients with ED-SCLC in a linear regression analysis. The MST difference was calculated as the difference in MST between the investigational and reference arms; the ORR difference was similarly defined. RESULTS: ORR difference between the treatment arms was modestly associated with the MST difference in the overall trials (R(2) = 0.3314). In contrast, the relationship was stronger among only trials in which prophylactic cranial irradiation was given to those having an objective response to the initial chemotherapy (R(2) = 0.6279). In this trial setting, large differences in ORR were needed to predict a survival advantage (1.2-day survival advantage per 2% increase in ORR). CONCLUSIONS: In the first-line treatment of ED-SCLC, a favourable relationship was detected between the two parameters in the selected trial setting. Large ORR differences were needed to predict a survival benefit, clearly suggesting the need for new chemotherapeutic agents.

Genetic analysis of Japanese patients with persistent hyperinsulinemic hypoglycemia of infancy: nucleotide-binding fold-2 mutation impairs cooperative binding of adenine nucleotides to sulfonylurea receptor 1.

To elucidate the genetic etiology of persistent hyperinsulinemic hypoglycemia of infancy (PHHI) in the Japanese population, we conducted a polymerase chain reaction-single-strand conformation polymorphism analysis of the sulfonylurea receptor 1 (SUR1) and Kir6.2 genes in 17 Japanese PHHI patients, including a pair of siblings from a consanguineous family. We also analyzed the glutamate dehydrogenase gene for the exons encoding an allosteric regulatory domain of the enzyme. In the SUR1 gene, we identified one frameshift (I446fsdelT) and two missense (R1420C, R1436Q) mutations. None of these mutations were found in control Japanese subjects. Siblings homozygous for the R1420C mutation had a mild form, whereas two patients heterozygous for the I446fsdelT and R1436Q mutations, respectively, exhibited a severe form of PHHI. Functional consequences of these mutations on K(ATP) function were evaluated using 86Rb+ efflux studies in COS-7 cells. SUR1-446fsdelT and SUR1-1436Q did not form a functional K(ATP). Western blot analysis after transient expression in COS-7 cells revealed the expression of SUR1-1436Q protein to be markedly reduced, suggesting SUR1-1436Q to be unstable in these cells. K(ATP)(SUR1-1420C) showed reduced responses to metabolic inhibition by oligomycin and 2-deoxyglucose. K(ATP) channels are under complex regulation by intracellular ATP and ADP. ATP both inhibits and activates these channels. The inhibition is probably mediated through direct ATP interaction with a pore-forming subunit Kir6.2, whereas the activation is likely to be through a regulatory subunit SUR1. There is a cooperative regulation of ATP and ADP binding to SUR1, and this cooperativity may be involved in regulating the K(ATP) channel. In SUR1-1420C, high-affinity binding of ATP to the nucleotide-binding fold (NBF)-1 was indistinguishable from that of wild-type SUR1. However, stabilization of ATP binding to NBF-1 by MgATP or MgADP was impaired, suggesting that this defect may account for impaired K(ATP)(SUR1-1420C) function. This is the first direct biochemical evidence that the cooperativity of nucleotide binding to SUR1 is impaired in a SUR1 mutant causing PHHI. No mutations were identified in the Kir6.2 and glutamate dehydrogenase genes. The genetic etiology of PHHI appears to be heterogeneous. SUR1 mutations may account for no more than 20% of PHHI cases in Japanese patients. Mutations of Kir6.2 and glutamate dehydrogenase genes are likely to be even less common.

Thrombopoietic activity of human interleukin-6.

Thrombopoietin (TPO), a regulatory factor in platelet production, was purified from the conditioned medium of TNK-01 cells cultured in the presence of human interleukin-1. The N-terminal sequence of purified TPO was determined to be VPPGEDSKDVAAPHRQPLT, identical to that of the N-terminal region of human interleukin-6 (IL-6). Two forms of TPO with molecular masses of 24 and 27 kDa were identified as IL-6 by Western analysis using an anti-IL-6 antibody. Commercial recombinant human IL-6 produced in Escherichia coli, stimulated megakaryocyte colony formation in the presence of mouse interleukin-3 and increased the number of peripheral platelets in mice in a dose-dependent manner. From these results, it is concluded that human IL-6 has thrombopoietic activity.

Frequency of provoked coronary vasospasm in patients undergoing coronary arteriography with spasm provocation test of acetylcholine.

This study examines the incidence of spasm by intracoronary injection of acetylcholine in Japanese patients who underwent coronary angiography. The subjects were 685 consecutive patients (477 men, mean age 63.2 +/- 7.5 years) who were studied with an acetylcholine test. Acetylcholine was injected in incremental doses of 20, 50, and 80 microg into the right coronary artery and 20, 50, and 100 microg into the left coronary artery. Spasm was defined as total or subtotal occlusion. Coronary vasospasm was determined in 221 patients (32.3%). Spasm occurred often during effort and rest in patients with angina (25 of 51, 49.0%), exertional angina (25 of 74, 33.8%), recent myocardial infarction (30 of 80, 37.5%), healed myocardial infarction (14 of 37, 37.8%), and especially in patients with rest angina (83 of 124, 66.9%), whereas spasm was relatively uncommon in patients with nonischemic heart disease (23 of 252, 9.1%). Spasm was superimposed on significant atherosclerotic lesions in 35.9% of patients as well as on nonfixed atherosclerotic lesions in 30.8% of patients. We conclude that >9% of Japanese patients may have coronary vasospasm with intracoronary injection of acetylcholine and recommend the provocation test for evaluating coronary vasospasm if coronary angiography is undertaken.

Major complications during spasm provocation tests with an intracoronary injection of acetylcholine.

This study sought to clarify major complications associated with acetylcholine testing. Serious major complications, such as sustained ventricular tachycardia, shock, and cardiac tamponade were determined in 4 of 715 patients (0.56%), but no cases of death or irreversible complications occurred. The spasm provocation test using acetylcholine should be performed carefully, although it is considered a safe and reliable method.

Usefulness of accelerated exercise following mild hyperventilation for the induction of coronary artery spasm : comparison with an acetylcholine Test.

STUDY OBJECTIVES: This study was performed to compare the results of accelerated exercise following mild hyperventilation and a standard acetylcholine (ACh) test for the induction of coronary artery spasm in patients with drug-induced coronary artery spasm. METHODS AND RESULTS: The subjects were 74 patients with angiographically confirmed coronary artery spasm who were examined using accelerated exercise (ie, exercise that was accelerated every minute according to the protocol of Bruce and Horsten) following mild hyperventilation and who were not receiving any medication. ACh was injected in incremental doses of 20 microg and 50 microg into the right coronary artery and incremental doses of 20 microg, 50 microg, and 100 microg into the left coronary artery. Positive coronary spasm was defined as > or =99% luminal narrowing. Accelerated exercise following a mild hyperventilation test was as useful for detecting evidence of ischemia as was an ACh test (48 patients [64.9%] vs 49 patients [66.2%], respectively; not significant). No difference was observed between ischemic changes on ECG as a result of the newly combined method and the occurrence of ACh-induced spasm. ACh-induced coronary vasospasm occurred in 61 patients (82.4%). In the remaining 13 patients, intracoronary administration of ergonovine provoked coronary spasms. No serious irreversible complications were detected as a result of this newly combined method. CONCLUSIONS: The effectiveness of our newly combined procedure is equivalent to that of an ACh test to diagnose patients with coronary artery spasm.

The continuous and simultaneous blood flow velocity measurement of four cerebral vessels and a peripheral vessel during cigarette smoking.

There has been no consensus about the acute effect of cigarette smoking on cerebral blood flow, and the continuous change of flow in four cerebral vessel flow with peripheral flow during different kinds of cigarette smoking has not been reported until now. Our results indicate smoking increases the flow of four cerebral vessels almost at the same time and with the same pattern. Many cerebral vessels began to show increases about 10 s after commencement. In most cases, cerebral blood velocity began to decrease between 10 and 20 s after cessation. Blood flow in peripheral vessels decreases after commencement, which is thought to be the effect of nicotine. The effect of high nicotine cigarettes is greater than that of low nicotine cigarettes. Continuous and simultaneous measurement of cerebral vessels by ultrasonic Doppler is though to be the only way to establish the detailed blood flow changes during smoking.

Bacteriolytic enzyme induced from pyocinogenic Pseudomonas aeruginosa. Purification and characterization of PR1-lysozyme.

A bacteriolytic enzyme, PR1-lysozyme, has been purified from the lysate of mitomycin C-induced pyocinogenic Pseudomonas aeruginosa, by acrinol treatment, Amberlite CG-50 chromatography, ammonium sulfate fractionation, Sephadex G-100 gel filtration and two cycles of SP-Sephadex C-50 chromatography. Homogeneity of the preparation was demonstrated by three electrophoretic techniques. PR1-lysozyme is a basic protein (pI, 9.4) and consists of a single polypeptide chain having a molecular weight of 24,000. The amino acid composition of the protein was analyzed, and no cystein residue was found among more than 210 amino acid residues. The optimum pH for enzymatic activity was 6.4 and the enzyme exhibited about 50 to 70 times greater specific activity than hen egg-white lysozyme when assayed with chloroform-killed P. aeruginosa as a substrate. By analyzing the products of enzymatic action on purified peptidoglycan of P. aeruginosa, the enzyme was identified as an N-acetylmuramidase, i.e., the same classification as hen-egg-white lysozyme. PR1-lysozyme did not show any activity towards intact cells of gram-positive and gram-negative bacteria tested. However, the enzyme was able to lyse chloroform-killed gram-negative and gram-positive bacteria.

Polypeptide and carbohydrate structure of recombinant human interleukin-6 produced in Chinese hamster ovary cells.

A glycosylated form of human interleukin 6 (hIL-6) has been produced in Chinese hamster ovary (CHO) cells transfected with a cDNA clone for human IL-6. Recombinant hIL-6 was purified from a culture supernatant of the transfected CHO cells, and used for structural characterization. The complete amino acid sequence, composed of 185 amino acid residues, was determined and is identical to that predicted from the cDNA sequence. However, a recombinant hIL-6 species lacking two amino acid residues (Ala-Pro) from the N-terminus was also found. Two disulfide bonds are formed, between Cys45 and Cys51 and between Cys74 and Cys84. Recombinant hIL-6 carries one O-linked carbohydrate chain, and Thr139 is fully O-glycosylated. A portion of recombinant hIL-6 protein carries one N-linked sialooligosaccharide chain, and the N-glycosylation occurs at Asn46. The structure of the N-linked sugar chains was estimated by a combination of sugar mapping and glycosidase digestion. The major structure of the N-linked sugar chain predicted was of a fucosylated biantennary or triantennary complex type. Fucosylated triantennary sugar chains with one or two N-acetyllactosaminyl repeats were also found. The structure of the O-linked sugar chain was determined by 500 mHz 1H-NMR to be NeuAc alpha 2-3Gal beta 1-3(NeuAc alpha 2-6) Gal-NAcol.

Physicochemical and biological comparison of recombinant human erythropoietin with human urinary erythropoietin.

Physicochemical and biological properties of recombinant human erythropoietin (rhEPO) were compared with human urinary erythropoietin (uEPO). uEPO and rhEPO were purified to apparent homogeneity from the urine of patients with aplastic anemia and from the conditioned medium of Chinese hamster ovary (CHO) cells transfected with a cDNA clone for human EPO, respectively. The microheterogeneous nature of both factors, observed on isoelectric focusing, is derived from the difference of the number of terminal sialic acid residues bound to the carbohydrate chains of the EPO molecule. The primary structure of rhEPO, consisting of 165 amino acid residues, was determined, and the C-terminal arginine predicted from the cDNA sequence was confirmed to be missing, as described previously (Recny et al. (1987) J. Biol. Chem. 262, 17156). Three N-glycosylation and one O-glycosylation sites of both factors were determined as Asn24, Asn38, and Asn83 and Ser126, respectively. Two disulfide linkages are located between Cys7 and Cys161, and between Cys29 and Cys33, in both EPOs. Hematogenic potencies of rhEPO and uEPO compared in normal and in partially nephrectomized rats were approximately the same. Both factors also stimulated the colony formation of CFU-E, BFU-E, and CFU-Meg in a dose-dependent manner. From these results, it is concluded that rhEPO produced in CHO cells transfected with cDNA clone for human EPO is indistinguishable from uEPO physicochemically and biologically, and is valuable for further research and for clinical use.

Structural characterization of natural and recombinant human granulocyte colony-stimulating factors.

Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein which stimulates predominantly neutrophilic granulocyte colony formation in mammals. Natural human G-CSF (hG-CSF) and recombinant hG-CSF produced in Chinese hamster ovary (CHO) cells transfected with the cDNA clone for hG-CSF have been purified to apparent homogeneity for structural and biological comparison. The amino acid sequence of recombinant hG-CSF, composed of 174 amino acid residues, was identical with that of natural hG-CSF and also with the sequence predicted from the cDNA. Both forms of hG-CSF have a free Cys-17 and two intramolecular disulfide linkages, between Cys-36 and Cys-42, and between Cys-64 and Cys-74. The O-glycosylation occurred at Thr-133 in both hG-CSFs. Similar CD spectra were obtained for both hG-CSFs. Additionally, both forms showed almost the same biological activities determined by in vitro colony-forming assay and in vivo assay. It is thus concluded that the recombinant hG-CSF is indistinguishable from its natural counterpart and that the former is valuable for more detailed characterization and clinical use.

Uptake of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and the N-methyl-4-phenylpyridinium ion (MPP+) into fetal mouse brain through the placenta.

The neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was found to be taken up into fetal mice through the placenta from a maternal mouse. C57 black mice were mated and MPTP (30 mg/kg) was given intramuscularly at the 18th day of gestation. Definite amounts of MPTP were detected in fetal brains by assay using high-performance liquid chromatography (HPLC) at 1 h after MPTP injection, and much higher contents of MPTP were found in maternal brains, too. An oxidative product of MPTP, the N-methyl-4-phenylpyridinium ion (MPP+), was also detected in brains of both mother and fetus and its concentrations in their brains were followed at 1, 3, 6, 12, 24 h after MPTP injection. The time to reach the maximal MPP+ concentration in brains was different between mother and fetus; 1 h and 3 h respectively. In addition to brain, considerable amounts of MPP+ were found in fetal liver, maternal liver and kidney, and in the placenta.

Positron emission tomography in juvenile Alexander disease.

A 13-year-old boy with cervical kyphosis was diagnosed as having juvenile Alexander disease because of the typical MRI findings, abnormally elevated alphaB-crystallin and heat shock protein 27 in the cerebrospinal fluid. Positron emission tomography with 18F-fluorodeoxyglucose demonstrated hypometabolism in the frontal white matter corresponding to the areas with leukodystrophy. However, the overlying gray matter preserved normal glucose metabolism.

Spontaneous remission of diabetes arrests progression of nephropathy in streptozotocin-treated spontaneously hypertensive rats.

Although antihypertensive therapy retards the progression of diabetic nephropathy associated with hypertension, it is not known whether glycemic control reverses or arrests diabetic nephropathy under untreated hypertension. We previously reported that spontaneous remission of diabetes occurred in the neonatal streptozotocin (STZ) model of spontaneously hypertensive rats (SHR) after 28 weeks of age, whereas hypertension persisted. Thus, we studied diabetic nephropathy before and after the recovery from hyperglycemia in this model. Two-day-old male SHR were injected intraperitoneally with STZ or vehicle for control. Hypertension was developed and maintained in both STZ and control groups in a similar degree. Before the amelioration of hyperglycemia, urinary albumin excretion increased progressively in STZ-treated SHR as compared with control (24 weeks; 1.6 +/- 0.5 mg/day, 17.5 +/- 2.3 mg/day, P < 0.001), and renal and glomerular hypertrophies were seen with mesangial expansion in STZ-treated SHR. However, along the recovery from hyperglycemia, urinary albumin excretion did not increase in the STZ-treated group, while it consistently increased in the control group (52 weeks; 25.4 +/- 10.0 mg/day, 29.7 +/- 11.4 mg/day, not significant). Furthermore, there were no significant differences in renal weight, glomerular tuft area, and the incidence of glomerular sclerosis between the two groups at 52 weeks of age. This study suggests that glycemic control may be effective for diabetic nephropathy even in the coexistence of untreated hypertension.

Effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration in prenatal stage on the dopamine system in the postnatal mouse brain.

A dopaminergic neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP), administered to a pregnant female was found to affect postnatally the catecholamine metabolism of the pups. MPTP (5 mg/kg body weight/day) was administered to pregnant C57 Black BYA mice daily for 7 days between the 12th and 18th day of gestation. Dopamine levels and tyrosine hydroxylase (TH) activity were measured in the whole brain from the pups sacrificed after birth. In MPTP-treated pups at 7 days of age, TH total activity (TH activity/brain) did not change (92% of the control value), while TH specific activity (TH activity/mg protein) was increased to 163% of that in control mice. Thus, TH homospecific activity (TH activity/mg TH protein) doubled compared to the control mice. At 28 days of age, both the total activity and the specific activity of TH in the brains of postnatal mice were reduced to 50% and 78% of the control, respectively. Dopamine concentration in the striatum was also reduced significantly. Reduction in the TH activity and dopamine concentration were also observed at the age of 12 weeks. These data suggest that the prenatal exposure to MPTP induced a prolonged reduction of TH activity in the brains of mice with a transient increase of TH homospecific activity during the postnatal period.

New non-invasive protocol for detection of coronary spastic angina with significant organic stenosis.

OBJECTIVES: This study sought to determine whether a newly-combined test, accelerated exercise following mild hyperventilation (HV) is more beneficial to detect ischaemic evidence in patients with pharmacology-induced coronary artery spasm (CAS) and luminal narrowing of > 75% than classic methods. METHODS AND RESULTS: Forty consecutive patients who all had luminal narrowing of > 75% but < 90% and pharmacology-induced coronary vasospasms of fixed lesions were involved in this study. In these patients, initial HV test, followed by treadmill (TM) exercise test and lastly the newly combined test were performed on three consecutive days. Of the 40 patients, firstly six, secondarily 16 and lastly 32 had positive responses to the HV test, TM exercise test, and newly combined test, respectively. The remaining six patients (15%) had negative results, although the triple sequential tests were performed. Thus, sensitivity of the HV test, the TM exercise test, and the newly combined test was 15% (6/40), 40% (16/40), and 84% (32/38), respectively. Specificity of the three tests were all 100% (46/46). Non-sustained ventricular tachycardia and hypotension were observed in two (5%) patients. However, no serious or irreversible complications were encountered in this study. CONCLUSIONS: We recommend the newly combined protocol rather than the classic tests for the detection of ischaemic evidence in patients with coronary spastic angina and fixed stenosis.

Continuous and simultaneous ultrasound Doppler velocimetry of the internal carotid and vertebral arteries: preliminary observations of cerebral blood flow changes with common carotid compression.

We have developed a new technique for simultaneous assessment of the four cervical cerebral arteries. Using a cervical brace with four freely adjustable Doppler probes attached, we studied four healthy young males and observed their cerebral blood flow changes during common carotid compression. The results indicated various reactivity in these subjects. We observed that carotid compression sometimes affects or stops the ipsilateral vertebral flow according to the intensity and location of compression. Also, the flow of the contralateral internal carotid artery (ICA) and vertebral artery (VA) showed remarkable increases greater than those due to ICA obliteration alone. This indicates the possibility that using only one probe during compression may often result in unknown obliteration of the ipsilateral VAs flow and could also produce other incomplete data. Moreover, with our technique, we would be able to assess the cerebral reserve more precisely and clearly in patients with carotid or vertebral occlusive diseases.