Haruhito Kikuchi

Do intronic mutations affecting splicing of WT1 exon 9 cause Frasier syndrome

The WT1 gene, one of the genes responsible for Wilms tumour, is thought to play a crucial role in the development of the kidneys and gonads. This gene encodes four protein isoforms resulting from two alternative splicing sites, one of which involves inclusion or exclusion of lysine, threonine, and serine (KTS) between the third and fourth zinc finger domains. WT1 is virtually always mutationally inactivated in patients with Denys-Drash syndrome. We analysed WT1 in eight patients who had been diagnosed as having this syndrome, and identified five previously unknown mutations affecting splicing donor sites of intron 9. These mutations affect alternative splicing. The isoforms retaining KTS are not produced. The clinical features of the patients with these intronic mutations were consistent with those of Frasier syndrome, characterised by a more slowly progressive nephropathy than Denys-Drash syndrome, associated streak gonads, and no Wilms tumour development. Our results indicate that WT1 isoforms, including/excluding KTS, have different functions in tumorigenesis and organogenesis of the kidneys and gonads.

Assessment of tailor-made prevention of atherosclerosis with folic acid supplementation: randomized, double-blind, placebo-controlled trials in each MTHFR C677T genotype.

AbstractThis study aimed at assessing the effect of folic acid supplementation quantitatively in each MTHFR C677T genotype and considered the efficiency of tailor-made prevention of atherosclerosis. Study design was genotype-stratified, randomized, double-blind, placebo-controlled trials. The setting was a Japanese company in the chemical industry. Subjects were 203 healthy men after exclusion of those who took folic acid or drugs known to effect folic acid metabolism. Intervention was folic acid 1 mg/day p.o. for 3 months. The primary endpoint was plasma total homocysteine level (tHcy). In all three genotypes, there were significant tHcy decreases. The greatest decrease was in the TT homozygote [6.61 (3.47-9.76) μmol/l] compared with other genotypes [CC: 2.59 (1.81-3.36), CT: 2.64 (2.16-3.13)], and there was a significant trend between the mutated allele number and the decrease. The tHcy were significantly lowered in all the genotypes, but the amount of the decrease differed significantly in each genotype, which was observed at both 1 and 3 months. Using these time-series data, the largest benefit obtained by the TT homozygote was appraised as 2.4 times compared with the CC homozygote. Taking into account the high allele frequency of this SNP, this quantitative assessment should be useful when considering tailor-made prevention of atherosclerosis with folic acid.

Constitutional WT1 correlate with clinical features in children with progressive nephropathy.

Editor—The WT1 tumour suppressor gene encodes a transcriptional factor containing four zinc fingers.1 2This gene has two alternative splicing regions, one consisting of 17 amino acids which are encoded by the whole of exon 5 and the other comprising three amino acids (lysine, threonine, and serine (KTS)) situated between the third and fourth zinc fingers encoded by the 3′ end of exon 9. Four isoforms of the gene thus occur depending on the presence or absence of these regions.3 These isoforms are present in a fixed proportion in tissues where they are expressed. WT1 is expressed from the condensing mesenchyme to mature podocytes in fetal kidneys. The other sites are genital ridges and fetal gonads. Therefore, this gene is thought to play an important role in the development of the kidneys and gonads.4 5Functional impairment of this gene is considered to give rise to urogenital abnormalities and Wilms tumours. Denys-Drash syndrome and Frasier syndrome, both of which are characterised by nephropathy with genital abnormalities, have been recognised as disorders related to WT1 mutations. Denys-Drash syndrome consists of the triad of progressive nephropathy characterised by diffuse mesangial sclerosis (DMS), genital abnormalities, and Wilms tumour.6 The incomplete form consists of nephropathy with genital abnormalities or Wilms tumour. In virtually all patients with Denys-Drash syndrome, point mutations are detected in the zinc finger domain encoded by exons 7 to 10 of the WT1 gene.7 The mutations noted in Denys-Drash syndrome patients are frequently missense changes in exons 8 and 9 that encode the second and third zinc fingers. Frasier syndrome is a clinical entity proposed by Moorthy et al 8 to be related to but distinguished from Denys-Drash syndrome. Frasier syndrome is characterised by a slowly progressing nephropathy, male pseudohermaphroditism, and no Wilms tumour. …

Nephrotic syndrome and end-stage renal disease with WT1 mutation detected at 3 years.

Abstract We report a boy who presented at 3 years with nephrotic syndrome and end-stage renal failure. Although histopathological findings showed end-stage kidney, isolated diffuse mesangial sclerosis (IDMS) was suspected because of his clinical course, and was confirmed by the presence of WT1 (Wilms tumor suppressor gene) mutation. He did not have ambiguous genitalia or Wilms tumor. The karyotype was 46:XY. A constitutional mutation in exon 7 (953G→A, 312Arg→Gin) was detected. A few cases of male IDMS, associated with WT1 mutations, have been reported. We believe that investigation for the WT1 mutation should be performed not only in Denys-Drash syndrome and IDMS, but also in end-stage renal disease with unexplained nephrotic syndrome of early onset. WT1 mutation-associated nephrotic syndrome has an increased risk of Wilms tumor. Careful ultrasound evaluations or bilateral nephrectomies are indicated.

A case of atypical POEMS syndrome without polyneuropathy

POEMS (Polyneuropathy, Organomegaly, Endocrinopathy, M‐protein, Skin changes) syndrome is a rare hematological disease associated with overproduction of pro‐inflammatory cytokines. Under the current nomenclature and diagnostic criteria for POEMS syndrome, the presence of characteristic polyneuropathy is required for diagnosis. We report a 43‐year‐old Japanese woman with organomegaly, endocrinopathy, M‐protein, skin lesions, as well as typical renal lesions and sclerotic bone lesions. Of note, neurological examinations and peripheral nerve conduction tests were normal in this patient. In view of the overwhelming number of otherwise characteristic signs and symptoms, we made a provisional diagnosis of ‘atypical POEMS syndrome without polyneuropathy’. If further similar cases are reported in the future, reconsideration of the nomenclature and/or diagnostic criteria for POEMS syndrome may be required.

Automated determination of drugs in serum by columnswitching high-performance liquid chromatography II. Separation of theophylline and its metabolites

The automated determination of theophylline and related compounds in human serum by column-switching high-performance liquid chromatography, including direct injection of serum samples, is described. TSK pre-column BSA-ODS and TSK gel ODS-80TM were used in the pre-column and analytical column, respectively. Serum samples of 20 microliters were directly injected on to the pre-column. After washing out serum proteins from the pre-column with 0.1 M NaH2PO4 at a flow-rate of 1.0 ml/min for 3.5 min, the effluent from the pre-column was introduced on to the analytical column by a column-switching device. The analysis was performed by stepwise gradient elution using 10 and 18% methanol in 0.1 M NaH2PO4. Theophylline and nine derivatives could be determined simultaneously within 40 min. The recovery of these compounds from serum was 95-103%. The linearity (1.0-50 micrograms/ml theophylline) and reproducibility (coefficient of variation less than 2.0%) were sufficient for drug monitoring at the lower and upper limits of therapeutic concentrations of theophylline.

Japanese guidelines of the management of hematuria 2013

Open image in new window Do hematuria standards differ by age or gender? [Statement] Red cell count distribution in urine varies with age and gender. However, since the significance of establishing hematuria standards for each group is unclear, more than 20 erythrocytes/µL or 5 erythrocytes/high power field (HPF) is considered to be the definition of hematuria. [Comment] Neither review articles discussing red cell count in the urine from healthy persons [1, 2] nor the American Urological Association (AUA)’s [3] Best Practice Policy concerning asymptomatic microscopic hematuria distinguish between age or gender in their definitions of hematuria. Moreover, Copley described that the normal number of red cells for children and adults is the same [2]. The standard of hematuria is globally defined as more than 5 erythrocytes/HPF in many reports [2, 4]. Japan’s own definitive text—the Standard Guideline for Urinary Sediment examination JCCLS GP1-P4—also states that below 4 erythrocytes/HPF are observed in the urine from healthy men and women [5]. Open image in new window What is the recommended urine collection method for hematuria diagnosis? [Statement] Intense exercise should be avoided before screening. Use of a clean container is recommended. Specification of urine sampling time, such as mid-stream urine, early morning first-void urine or random sample, is also recommended. (Recommendation grade A) [Comment] The Japanese Committee for Clinical Laboratory Standardization (JCCLS) has issued two documents concerning routine urinalysis: Proposed Guideline for the Urinary Reagent Method [1] and Standard Guideline for Urinary Sediment [2]. Generally, a midstream urine sample is used and physical exercise before collection of urine should be avoided as it may cause hematuria or hemoglobinuria. Open image in new window Do urine blood test strips produced by various manufacturers differ in their sensitivity? [Statement] Although data sheets indicate no differences in sensitivity among various urine test strips, some differences were observed when measuring urine samples. [Comment] The Japanese Committee for Clinical Laboratory Standards (JCCLS) defined that 1+ of a urine blood reagent strip should correspond to 0.06 mg/dL of hemoglobin or about 20 erythrocytes/µL in 2004. However, the upper and/or lower limits of 1+ and other ranks (2+ , 3+ , etc.) have not been standardized. Results of a JCCLS investigation using pooled urine samples spiked with hemoglobin showed considerable differences among test strip manufacturers. Open image in new window What is the recommended urine collection method for medical checkup? [Statement] Adding to the methods described in CQ2, 1) utilizing early morning first-void and mid-stream urine and 2) refraining from ingestion of foods rich in ascorbic acid (vitamin C) are recommended. (Recommendation Grade B) [Comment] The fundamentals of urine collection do not change for medical checkups. However, in medical checkups for schoolchildren, collection of early morning first-void urine is indicated to avoid hematuria caused by physical exercise. Since the urine test strip for blood uses the peroxidase-like action of hemoglobin, if a substance with a reduction action exists, it will yield a false negative result. Therefore ingestion of foods rich in ascorbic acid (vitamin C), which is the most frequent reducing substance appearing in urine, should be avoided the night preceding a checkup. Open image in new window Can you distinguish glomerular hematuria from non-glomerular hematuria by morphological analysis of urinary erythrocytes? [Statement] There are various shapes and sizes of urinary erythrocytes in hematuria. Information pertaining to urinary erythrocyte morphology is therefore useful in deciding the origin of hematuria. Even if glomerular hematuria is morphologically suspected, urinary tract disease other than glomerular disease cannot be ruled out. Moreover, not all types of hematuria can be properly classified. As results of the morphological discrimination method vary among examiners, it is recommended that screening follow the Standard Guideline for Urinary Sediment Examination [Japanese Committee for Clinical Laboratory Standard (JCCLS), Document GP1-P4, Proposed Guideline], which is the standard examination method in Japan. [Comment] In non-glomerular hematuria such as hematuria originated from the lower urinary tract, urinary erythrocytes take the form of atrophy or disc and their morphologies are nearly identical although varying somewhat in size. Furthermore, they are most likely to be rich in hemoglobin. Such erythrocytes are called isomorphic RBCs (glomerular type RBCs1). In contrast, erythrocytes in glomerular hematuria often present with various forms of casts, including erythrocyte casts and proteinuria. These erythrocytes most often appear in various forms, such as a doughnut shape with humps or a target shape, in the same specimen, and are called dysmorphic RBCs (non-glomerular type RBSs) (see Footnote 1). Erythrocyte morphology is generally observed and evaluated at the time of urinary sediment examination using a light microscope in Japan, whereas it is often done using a phase-contrast microscope in other countries. Facilities increasingly use a urinary formed-element analyzer using flow cytometry. None of these methods are capable of classifying all types of hematuria. Therefore, non-glomerular hematuria cannot be ruled out even when glomerular hematuria is morphologically suspected. Open image in new window What are the differences between dysmorphic type RBC2 and isomorphic type RBC (see Footnote 2) in the information about urinary erythrocyte morphology acquired by an autoanalyzer? [Statement] In Japan, urinary components that are observed by an autoanalyzer are distinguished as information pertaining to urinary formed elements from the results of urinary sediment examination. A urinary formed-element analyzer using cytometry can discriminate among blood cells, epithelial cells, casts and other formed elements by processing scattered light from particles as well as light signals corresponding to fluorescence intensity. Urinary erythrocytes can be distinguished based on the bias of the particle size distribution curve of discriminated erythrocytes. Taking advantage of the feature that erythrocytes from the glomeruli are generally smaller than those from other sites, it is possible to classify erythrocytes into dysmorphic and isomorphic type RBC depending on shifts in particle size toward the smaller and larger sides, respectively. Making the above classification may not always be possible, as hematuria specimens may contain a small number of erythrocytes, urine properties such as high acid content may be present, or urine may be very hypotonic or the specimen in a poor state of preservation following collection. Additionally, there may be a mixed dysmorphic/isomorphic RBC type. [Comment] The measurement principle of the urinary formed-element analyzer using cytometry is that particle elements are displayed on a scattergram for analysis by measuring their scattering light and fluorescence provoked by laser light after they are stained with fluorochrome for exact characterization of their sizes, shapes and nuclei. This analytical method can characteristically depict a clear distribution of broadly-divided particle elements (a particle size distribution pattern), although it has limited ability for minute classification of elements other than erythrocytes and leukocytes. Morphological observation of urinary erythrocytes is usually made with urinary sediment examination in Japan. However, there is a major flaw in this examination, which employs microscopic observation of urinary sediment acquired by urine centrifugation, due to cellular element destruction caused by centrifugation, supernatant residual erythrocytes and apparent low measurement values in the hypotonic urine. In contrast, the cytometry-based urinary formed-element analyzer, which analyzes non-centrifuged urine, is superior in quantitative performance. Moreover, it presumably can distinguish dysmorphic RBCs in a glomerular hematuria specimen, which often has variously-formed erythrocytes (humped doughnut- and target-shaped, for example) because these dysmorphic RBCs often fall on the smaller size side of the particle size distribution due to their tiny volume resulting from hemoglobin loss. Nevertheless, this analyzer does not always clearly distinguish urine specimens due to too few urinary erythrocytes, properties such as high acidity or hypotonicity, or their composite components.

High-Dose Hook Effect in an Immunochromatography–Optical Quantitative Reader Method for Myoglobin

Triaging of patients with chest pain to rule out acute myocardial infarction is important. Myoglobin is thought to be an effective marker for this purpose in the early hours after the onset of symptoms because it is released into the blood shortly after myocardial damage (1)(2)(3). The Cardiac Reader (Roche) provides a platform for the quantitative immunologic measurement of whole-blood myoglobin. The measuring range of the Cardiac Reader for myoglobin is 30–700 μg/L, and the upper reference limit is 80 μg/L. In a previous study, the within-series CV (the mean value of 20 within-run CVs with 20 instruments) was 5–10% and was almost constant throughout the measuring range (4). The assay system is thought to be suitable for near-patient use because of its easy operation and the short assay time. We observed two cases whose blood myoglobin concentrations showed falsely low values with this method. Case A was an 84-year-old male in cardiogenic …

Serum alcohol dehydrogenase: A sensitive biomarker of ongoing graft function after liver transplantation

Abstract:  Alcohol dehydrogenase (ADH) is an enzyme specifically located in the cytoplasm of hepatocytes. The purpose of this study was to assess the potential usefulness of serum ADH activity as a biomarker of graft function following liver transplantation. Blood samples were obtained from 26 patients who underwent living‐donor liver transplantation. In patients without any post‐operative complication, serum ADH activity normalized at 2.9 ± 1.2 d. Values of serum ADH activity were remarkably elevated in patients with vascular complications, whereas they were only slightly elevated or remained within the reference range in patients with acute cellular rejections. In vascular complications, serum ADH activity peaked prior to elevation of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and once the cause of damage was resolved, the values returned to reference range more quickly than did ALT and AST. In conclusion, monitoring serum ADH activity in addition to ALT and AST may provide more sensitive ongoing graft status and valuable information for the differential diagnosis of vascular complications and acute cellular rejection.