Genetic and phenotypic profile of 112 patients with X-linked Charcot-Marie-Tooth disease type 1.

BACKGROUND AND PURPOSE: X-linked Charcot-Marie-Tooth disease type 1 (CMTX1), caused by mutations in gap junction protein beta 1 (GJB1), is characterized by various central nervous system symptoms and gender differences of clinical severity. The aim of this study was to identify the frequency and mutation spectrum of CMTX1 patients in Japan and to demonstrate their phenotypic diversities. METHODS: Using three high-throughput sequencing systems, targeted gene panel sequencing on 1483 unrelated index patients with suspected Charcot-Marie-Tooth (CMT) disease was performed. The peripheral and central nervous system involvements of all patients with GJB1 variants were assessed retrospectively and a detailed gender comparison was conducted with the CMT examination score. RESULTS: Twenty-three novel and 36 described GJB1 variants were identified from 88 pedigrees, in which 34 female and 78 male patients were enrolled. Mean age at onset of the male patients was much younger than the females, 21.56 ± 17.63 years vs. 35.53 ± 23.72 years (P = 0.007). Male patients presented with more severe phenotypes in every examination item, but statistical differences were observed only in motor dysfunctions of the lower extremities and vibration sensation. No significant sensory difference was identified between genders, either clinically or electrophysiologically. Central nervous system dysfunctions were found in 15 patients from 12 pedigrees. Therein, six patients developed stroke-like phenotypes, with dysarthria as the leading symptom. CONCLUSIONS: A relatively lower frequency of CMTX1 (5.9%) was demonstrated and a broad mutation spectrum of GJB1 was described. Detailed clinical differences between genders and various central nervous system symptoms were also illustrated, even in the same pedigree.

WNK1/HSN2 founder mutation in patients with hereditary sensory and autonomic neuropathy: A Japanese cohort study.

The clinical and genetic spectrum of hereditary sensory and autonomic neuropathy (HSAN) is still unknown in Japan. We collected a broad cohort of 33 unrelated patients with predominant sensory and/or autonomic dysfunctions, who were referred to our genetic laboratory. A gene panel sequencing targeting 18 HSAN-related genes was performed using a next-generation sequencing system. A recurrent frame shift mutation in the WNK1/HSN2 gene, c.3237_3238insT (p.Asp1080*), was detected in 5 patients. This mutation was homozygous in 4 cases and of a compound heterozygous genotype in 1 case. Geographic and haplotype analysis of all 5 patients suggested a founder event. In addition, a novel heterozygous nonsense variant, c.2615C>G (p.Ser872*), was identified. All the 5 patients presented with severe sensory and autonomic dysfunctions at birth or during adolescence. In 2 patients, an uncommon phenotype of acute pathological pain presented at ~50 years of age. Here, we present the first founder mutation of WNK1/HSN2, in addition to French Canadian, which accounts for ~15.2% of Japanese patients with HSAN in our cohort. We have also reviewed all previously described mutations in WNK1/HSN2 and reconciled their nomenclature strategy on the basis of the current longest transcript.

Clinical and mutational spectrum of Japanese patients with Charcot-Marie-Tooth disease caused by GDAP1 variants.

BACKGROUND: Mutations in GDAP1 are responsible for heterogeneous clinical and electrophysiological phenotypes of Charcot-Marie-Tooth disease (CMT), with autosomal dominant or recessive inheritance pattern. The aim of this study is to identify the clinical and mutational spectrum of CMT patients with GDAP1 variants in Japan. MATERIALS AND METHODS: From April 2007 to October 2014, using three state-of-art technologies, we conducted gene panel sequencing in a cohort of 1,030 patients with inherited peripheral neuropathies (IPNs), and 398 mutation-negative cases were further analyzed with whole-exome sequencing. RESULTS: We identified GDAP1 variants from 10 patients clinically diagnosed with CMT. The most frequent recessive variant in our cohort (5/10), c.740C>T (p.A247V), was verified to be associated with a founder event. We also detected three novel likely pathogenic variants: c.928C>T (p.R310W) and c.546delA (p.E183Kfs*23) in Case 2 and c.376G>A (p.E126K) in Case 8. Nerve conduction study or sural nerve biopsy of all 10 patients indicated axonal type peripheral neuropathy. CONCLUSION: We identified GDAP1 variants in approximately 1% of our cohort with IPNs, and established a founder mutation in half of these patients. Our study originally described the mutational spectrum and clinical features of GDAP1-related CMT patients in Japan.

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.

MRI depiction and 3D visualization of three anterior cruciate ligament bundles.

The anterior cruciate ligament (ACL) is divided into three fiber bundles (AM-M: anteromedial-medial, AM-L: anteromedial-lateral, PL: posterolateral). We attempted to depict the three bundles of the human ACL on MRI images and to obtain 3-dimensional visualization of them. Twenty-four knees of healthy volunteers (14 males, 10 females) were scanned by 3T-MRI using the fat suppression 3D coherent oscillatory state acquisition for the manipulation of imaging contrast (FS 3D-COSMIC). The scanned images were reconstructed after the isotropic voxel data, which allows the images to be reconstructed in any plane, was acquired. We conducted statistical examination on the identification rate of the three ACL bundles by 2D planes. Segmentation and 3D visualization of the fiber bundles using volume rendering were performed. The triple-bundle ACL was best depicted in the oblique axial plane. While the AM-M and AM-L bundles were clearly depicted in all cases, the PL bundle was not clearly visualized in two knees (8%). Therefore, the three ACL bundles were depicted in 22 knees (92%). The results of 3D visualization of the fiber arrangement agreed well with macroscopic findings of previous anatomical studies. 3T-MRI and the isotropic voxel data from FS 3D-COSMIC made it possible to demonstrate the identifiable depiction of three ACL bundles in nearly all cases. 3D visualization of the bundles could be a useful tool to understand the ACL fiber arrangement. Clin. Anat. 30:276-283, 2017. 2016 The Authors. Clinical Anatomy published by Wiley Periodicals, Inc. on behalf of American Association of Clinical Anatomists.

Schimke immuno-osseous dysplasia: SMARCAL1 loss-of-function and phenotypic correlation.

BACKGROUND: Schimke immuno-osseous dysplasia (SIOD) is an autosomal recessive pleiotropic disorder caused by mutations in SMARCAL1. SMARCAL1 encodes an enzyme with homology to the SNF2 chromatin remodelling proteins. METHODS: To assess the affect of SMARCAL1 mutations associated with SIOD on SMARCAL1 expression and function, we characterised the effects of various mutations on mRNA and protein expression in patient tissues and cell lines, and the ATPase activity, subcellular localisation, and chromatin binding of SMARCAL1 missense mutants. RESULTS: The SIOD associated SMARCAL1 mutations affected SMARCAL1 protein expression, stability, subcellular localisation, chromatin binding, and enzymatic activity. Further, expressing SMARCAL1 missense mutants in Drosophila melanogaster showed that disease severity was inversely proportionate to overall SMARCAL1 activity. CONCLUSION: Our results show for the first time that SMARCAL1 binds chromatin in vivo and that SIOD arises from impairment of diverse SMARCAL1 functions.

The sentinel node concept in prostate cancer: Present reality and future prospects.

A sentinel node (SN) is defined as the first site where cancer cells are carried by lymph flow from a tumor. If this definition (SN concept) correctly reflects the clinical reality, intraoperative SN biopsy would facilitate precise nodal staging. In malignant melanoma, a prolonged survival has been evidenced by a large-scale randomized controlled study. On the contrary, research on SN concept in deeply located cancers including prostate cancer, is still investigative, and no concrete data from clinical trials are yet available. Since 1993, several investigators have demonstrated that the SN concept could be applied in prostate cancer patients as well with high accuracy. Although promising and technically feasible in pre-clinical settings, many hurdles remain to be cleared before clinical application can be recommended. This review addresses the current status and related issues of the SN concept in prostate cancer, and discusses the future directions.

Bradykinin stimulates the release of tissue plasminogen activator in human coronary circulation: effects of angiotensin-converting enzyme inhibitors.

OBJECTIVES: The goal of this study was to determine: 1) whether bradykinin (BK) directly stimulates tissue plasminogen activator (tPA) secretion in human coronary circulation, and 2) whether angiotensin-converting enzyme (ACE) inhibition favorably alters the fibrinolytic balance regulated by BK. BACKGROUND: Bradykinin is a potent stimulator of tPA secretion in endothelial cells; however, the effect of BK on tPA release in the human coronary circulation has not been studied. METHODS: Fifty-six patients with atypical chest pain were randomly assigned to two groups: 25 patients were treated with the ACE inhibitor enalapril (ACE inhibitor group), and 31 were not treated with ACE inhibitors (non-ACE inhibitor group). Graded doses of BK (0.2, 0.6, 2.0 microg/min), acetylcholine (ACh) (30 microg/min) and papaverine (PA) (12 mg) were administered into the left coronary artery. Coronary blood flow (CBF) was evaluated by Doppler flow velocity measurement. Blood samples were taken from the aorta (Ao) and the coronary sinus (CS). RESULTS: Bradykinin induced similar increases in CBF in both groups. The net tPA release induced by BK was dose-dependently increased in both groups, and the extent of that increase in the ACE inhibitor group was greater than that in the non-ACE inhibitor group. Bradykinin did not alter plasminogen activator inhibitor-1 (PAI-1) levels in the Ao or CS in either group. Neither ACh nor PA altered tPA levels or PAI-1 levels in either group. CONCLUSIONS: Intracoronary infusion of BK stimulates tPA release without causing any change in PAI-1 levels in the human coronary circulation. In addition, this effect of BK is augmented by an ACE inhibitor.

Barriers to providing diabetes care in community health centers.

OBJECTIVE: We aimed to identify barriers to improving care for individuals with diabetes in community health centers. These findings are important because many such patients, as in most other practice settings, receive care that does not meet evidence-based standards. RESEARCH DESIGN AND METHODS: In 42 Midwestern health centers, we surveyed 389 health providers and administrators about the barriers they faced delivering diabetes care. We report on home blood glucose monitoring, HbA1c tests, dilated eye examinations, foot examinations, diet, and exercise, all of which are a subset of the larger clinical practice recommendations of the American Diabetes Association (ADA). RESULTS: Among the 279 (72%) respondents, providers perceived that patients were significantly less likely than providers to believe that key processes of care were important (overall mean on 30-point scale: providers 26.8, patients 18.2, P = 0.0001). Providers were more confident in their ability to instruct patients on diet and exercise than on their ability to help them make changes in these areas. Ratings of the importance of access to care and finances as barriers varied widely; however, >25% of the providers and administrators agreed that significant barriers included affordability of home blood glucose monitoring, HbA1c testing, dilated eye examination, and special diets; nonproximity of ophthalmologist; forgetting to order eye examinations and to examine patients' feet; time required to teach home blood glucose monitoring; and language or cultural barriers. CONCLUSIONS: Providers in health centers indicate a need to enhance behavioral change in diabetic patients. In addition, better health care delivery systems and reforms that improve the affordability, accessibility, and efficiency of care are also likely to help health centers meet ADA standards of care.

Stem cell factor protects c-kit+ human primary erythroid cells from apoptosis.

OBJECTIVE: It has been reported that stem cell factor (SCF) promotes cell survival in primary cultured human erythroid colony-forming cells (ECFC). Given the heterogeneous nature of ECFC, which may affect interpretation of the data, we purified c-kit+ ECFC and investigated the specificity and mechanisms of the anti-apoptotic effects of SCF on these cells. MATERIALS AND METHODS: Glycophorin A+ (GPA+) c-kit+ cells were purified from primary cultured ECFC derived from purified human CD34+ cells. The GPA+c-kit- and nonerythroid cells were generated from the same CD34+ cells. Apoptosis of ECFC was investigated in the absence or presence of SCF and erythropoietin (EPO) in serum-free medium. DNA fragmentation was measured with enzyme linked immunosorbent assay for oligonucleosome-sized DNA, gel electrophoresis, and annexin V labeling. Characterization of expanded cells and enriched cells was performed using multiparameter flow cytometry. For Akt assay, cells were lysed and the cleared lysates subjected to SDS-PAGE followed by Western blotting. RESULTS: In GPA+c-kit+ cells, deprivation of cytokine caused rapid DNA fragmentation within 4 hours that reached a maximum at 6 hours. This was partially but clearly prevented by SCF or EPO. In contrast, no significant DNA fragmentation was seen in GPA+c-kit- and nonerythroid cells within 24 hours. PP2, a specific Src family kinase inhibitor, but not its inactive analogue PP3, reversed the anti-apoptotic effects of SCF. PP2 also inhibited SCF-induced phosphorylation of Akt. CONCLUSION: These data indicate that SCF protects purified human GPA+c-kit+ cells from apoptosis and suggest that kit-mediated Src kinase activation is involved in Akt activation and cell survival.

Assessment of MRI criteria for MS in Japanese MS and HAM/TSP.

We evaluated the usefulness of the MRI criteria for multiple sclerosis (MS) proposed by Paty et al and Fazekas et al in 36 Japanese MS patients, using HTLV-I-associated myelopathy (HAM)/tropical spastic paraparesis (TSP) as the control. Although 30 of 36 HAM/TSP patients had multiple white matter lesions on T2-weighted cranial MRI, only two fulfilled the MRI criteria for MS. At the same time, 31 of the 36 MS patients fulfilled the primary MRI criterion, yielding 93% specificity and 86% sensitivity for the criterion. MS has disease-specific MRI abnormalities.

Lewy body-type degeneration in cardiac plexus in Parkinson's and incidental Lewy body diseases.

Heart tissues of patients with PD or incidental Lewy body (LB) disease (ILBD) were examined by light and electron microscopy. LBs and alpha-synuclein-positive neurites were identified in the hearts from 9 of 11 patients with PD and from 7 of 7 patients with ILBD. LBs were present in both tyrosine hydroxylase-positive and -negative nerve processes, which are nerves of extrinsic sympathetic and intrinsic origin, respectively. These findings provide histologic evidence that the postganglionic sympathetic and intrinsic neurons in the heart are involved in the PD disease process.

A novel MPZ gene mutation in dominantly inherited neuropathy with focally folded myelin sheaths.

We found the association of a heterozygous novel MPZ gene point mutation, Ile62Phe in exon 2, with autosomal dominant motor and sensory neuropathy with focally folded myelin sheaths. Family study revealed that de novo Ile62Phe mutation on the MPZ gene occurred in the proband and was inherited by her children with early onset slowly progressive neuropathy. Our study suggests that the characteristic pathologic findings of the sural nerve in these patients are closely related to the site and nature of amino acid substitutions of the MPZ gene.

Hereditary motor and sensory neuropathy with myelin folding and juvenile onset glaucoma.

OBJECTIVE: We describe three patients from a family with motor and sensory neuropathy accompanied by open-angle glaucoma. BACKGROUND: Autosomal recessive demyelinating hereditary motor and sensory neuropathies (HMSN) include different disorders. To our knowledge, autosomal recessive HMSN has not been associated with juvenile onset glaucoma. METHODS: Sural nerve pathology of the three patients were examined, and genetic analysis of the family was performed. RESULT: - The most prominent pathologic finding was a highly unusual myelin abnormality consisting of irregular redundant loops and folding of the myelin sheath. The family survey supports autosomal recessive inheritance. The molecular analysis failed to demonstrate either linkage of the disease to MPZ gene, PMP22 gene, Cx32 gene, orEGR2 gene. Analysis did not establish linkage of the disease to the locus of CMT4A, 4B, and 4C genes. CONCLUSION: The present cases may represent a new type of HMSN accompanied by juvenile onset glaucoma.

Induction of angiotensin converting enzyme and angiotensin II receptors in the atherosclerotic aorta of high-cholesterol fed Cynomolgus monkeys.

Antiatherogenic effects of imidapril and involvement of renin angiotensin system were examined in experimental atherosclerosis induced by feeding a high-cholesterol diet to Cynomolgus monkeys. Eighteen male monkeys were divided into three groups and placed under (1) normal diet (normal group), (2) high-cholesterol diet (control group), (3) high-cholesterol diet with imidapril (20 mg/kg body wt/day, orally) treatment (imidapril group). At the end of the experiment, the normal group showed no apparent atherosclerosis in their aorta evaluated by oil red-O staining, while the control group exhibited marked atherosclerotic involvement of the intimal surface of the aorta (58.4 +/- 9.3%, P < 0.01). Imidapril reduced systolic blood pressure and atherosclerotic involvement (24.1 +/- 5.5%, P < 0.05). Total cholesterol content of the descending thoracic aorta was also significantly reduced in the imidapril group. In the atherosclerotic vessels, angiotensin converting enzyme (ACE) activity evaluated by quantitative in vitro autoradiography was significantly increased in the intimal lesion. Further evaluation revealed angiotensin II (Ang II) type I (AT1) receptor density was significantly increased in the medial lesion and type II (AT2) receptor density in the adventitia. When the progression of atherosclerosis was impeded by imidapril treatment, the ACE activity level as well as the AT1 and AT2 receptor density remained at normal. Expression of mRNA for fibronectin, TGF-beta1, types I and III collagen was studied by Northern blot analysis. No significant differences in types I and III collagen mRNA levels were found between the control and imidapril group. On the other hand, mRNA expression for fibronectin and TGF-beta1 were much lower in the imidapril group than in the control group. These results suggest that increased production of Ang II and activated receptors may be involved in atherosclerotic process in this model and also antiatherogenic effect of imidapril may be derived from reduction of local Ang II production as well as its hypotensive action.

Active specific immunotherapy using the immune reaction of a low-dose irradiated tumor tissue.

Active specific immunotherapy using the immune reaction of a low-dose irradiated tumor tissue was studied on the transplanted MM46 tumor of female C3H/He mice after radiotherapy. MM46 tumor cells were inoculated into the right hind paws of mice. On the 5th day, irradiation with the dose of 3000 rad was performed. On the 11th day, tumor cells and mononuclear cells, which were separated from the low-dose irradiated tumor tissue (2000 rad on the fifth day), were injected into the left hind paws of the tumor-bearing mice. Effectiveness of this active specific immunotherapy against tumor was evaluated by the regression of tumor and survival rate of mice. Tumor was markedly regressed and survival rate was significantly increased by the active specific immunotherapy.

Gene for hereditary motor and sensory neuropathy (proximal dominant form) mapped to 3q13.1.

Hereditary motor and sensory neuropathy with proximal dominant involvement (HMSN-P) has been reported as a new type of HMSN with the disease gene locus in the 3p14.1-q13 region. To further narrow down the gene locus, we performed fine linkage mapping using the linkage disequilibrium method. Analysis of DNA marker haplotypes and genetic cross-over sites showed the disease gene locus to be in the 3.1 cM interval bracketed by D3S1591 and D3S1281. Linkage disequilibrium analysis with DISMULT using 9 marker loci jointly in this region showed a lod score of 4.93 (P < 0.00000095). Consequently, the HMSN-P gene almost certainly lies on chromosome 3q13.1 and shows evidence of linkage disequilibrium.

Germline mosaicism of MPZ gene in Dejerine-Sottas syndrome (HMSN III) associated with hereditary stomatocytosis.

We report on two sisters with Dejerine-Sottas syndrome (DSS) who had a heterozygous Gly 167 Arg mutation in the myelin protein zero (MPZ) gene and hereditary stomatocytosis (HSt). Genetic haplotype analysis suggested that the allele with the MPZ gene mutation originated from maternal lineage. However, the parents, who were normal clinically and electrophysiologically, had no mutation in the MPZ gene. Therefore, the MPZ gene mutation in these sisters was due to germline mosaicism of the MPZ gene in their mother. Stomatocytosis was detected in their mother and a sister who had no neurological symptoms, and therefore autosomal dominant HSt was suspected in this family. As stomatocytosis is very severe in our patients with DDS, we speculate that the association of DSS with stomatocytosis is coincidental but may have additively affected erythrocyte morphology. To our knowledge, these are the first familial cases of DSS with a mutation due to germline mosaicism of the MPZ gene to be reported.

Synthesis and antiviral activity of deoxy analogs of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) as potent and selective anti-HIV-1 agents.

The effect of substitution in the acyclic structure of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)-thymine (HEPT) on anti-HIV-1 activity was investigated by synthesizing a series of deoxy analogs and related compounds. Preparation of 1-[(2-alkyloxyethoxy)methyl]-6- (phenylthio)thymine (2-4) derivatives was carried out based on alkylation of HEPT with primary alkyl halides. Preparation of the 1-[(alkyloxy)methyl]-6-(phenylthio)thymine (26-31) and 1-[(alkyloxy)methyl]-6-(arylthio)-2-thiouracil (32-45) derivatives was carried out on the basis of LDA lithiation of 1-[(alkyloxy)-methyl]thymine (9-14) and 1-[(alkyloxy)methyl]-2-thiouracil (15-25) followed by reaction with diaryl disulfides. The oxidative hydrolysis of the 2-thiouracil derivatives gave 1-[(alkyloxy)methyl]-6-(arylthio)uracil derivatives (46-57). 1-Alkyl-6-(phenylthio)thymine (59-61) derivatives were prepared on the basis of alkylation of 6-(phenylthio)thymine (58). Methylation of the hydroxyl group of HEPT did not affect the anti-HIV-1 activity of HEPT. Substitution of the 1-(2-hydroxyethoxy)methyl group by ethyl, butyl, methoxymethyl, (propyloxy)methyl, and (butyloxy)-methyl groups somewhat improved the original anti-HIV-1 activity of HEPT. Substitution with ethoxymethyl and (benzyloxy)methyl groups further potentiated the activity [EC50: 1-(ethoxy-methyl)-6-(phenylthio)thymine (27), 0.33 microM; 1-[(benzyloxy)methyl]-6-(phenylthio)thymine (31), 0.088 microM]. When the 5-methyl group of 27 and 31 was replaced by an ethyl or an isopropyl group, the anti-HIV-1 activity was improved remarkably [EC50: 5-ethyl-1-(ethoxymethyl)-6-(phenylthio)-uracil (46), 0.019 microM; 5-ethyl-1-[(benzyloxy)methyl]-6-(phenylthio)uracil (52), 0.0059 microM; 5-isopropyl-1-(ethoxymethyl)-6-(phenylthio)uracil (55), 0.012 microM; 5-isopropyl-1-[(benzyloxy)methyl]-6-(phenylthio)uracil (56), 0.0027 microM]. Introduction of two m-methyl groups into the phenylthio ring also potentiated the activity.