Toyomi Kamesaki

An intronic variable number of tandem repeat polymorphisms of the cold-induced autoinflammatory syndrome 1 ( CIAS1) gene modifies gene expression and is associated with essential hypertension

Cold-induced autoinflammatory syndrome 1 (CIAS1) gene is a member of the NALP subfamily of the CATERPILLER protein family that is expressed predominantly in peripheral blood leukocytes, which is to regulate apoptosis or inflammation through the activation of NF-κB and caspase. Recent genetic analyses suggested an association between inflammation and oxidative stress-related genes in the development of hypertension. This is the first genetic study indicating an association between the CIAS1 gene and susceptibility to essential hypertension (EH). The frequency of subject with the homozygote of 12 repeat allele was significantly higher in patients with hypertension compared with control subjects (987 cases, 924 controls) (P=0.030; odds ratio=1.24) at a novel VNTR polymorphism of CIAS1 intron 4 loci. We also found that the mean of systolic blood pressure of homozygotes of 12 repeat allele was 6.4 mmHg higher than those of homozygotes of non-12 repeat allele in male random population (P=0.009). The frequency of six SNPs spanning of the CIAS1 gene was not significantly between patients and controls. The real-time PCR analysis showed that among healthy young adults, 12-12 subjects expressed CIAS1 mRNA in peripheral leukocytes significantly more abundantly than homozygote of non-12 repeat alleles subjects (P<0.05). Reporter gene assay of the CIAS1-VNTR in HL60 stimulated by lipopolysaccharides showed that the intronic sequence involving 12 repeat increased the expression of luciferase compared with 9, 7, and 6 repeats. Thus, we propose here the CIAS1 is associated with EH through the dominant expression of transcripts, which may depend on the CIAS1-VNTR genotype.

Cut-off value of red-blood-cell-bound IgG for the diagnosis of Coombs-negative autoimmune hemolytic anemia.

Direct antiglobulin test (DAT)‐negative autoimmune hemolytic anemia (Coombs‐negative AIHA) is characterized by laboratory evidence of in vivo hemolysis, together with a negative DAT performed by conventional tube technique (CTT) in clinically suspected AIHA patients. The immunoradiometric assay (IRMA) for red‐blood‐cell‐bound immunoglobulin G (RBC‐IgG) can be used to diagnose patients in whom CTT does not detect low levels of red cell autoantibodies. We investigated the diagnostic cutoff value of the IRMA for RBC‐IgG in Coombs‐negative AIHA and calculated its sensitivity and specificity. Of the 140 patients with negative DAT by CTT referred to our laboratory with undiagnosed hemolytic anemia, AIHA was clinically diagnosed in 64 patients (Coombs‐negative AIHA). The numbers of Coombs‐negative AIHA and non‐AIHA patients changed with age and gender. The cutoff values were determined from receiver operating characteristic (ROC) curve according to age and gender. The IRMA for RBC‐IgG proved to be sensitive (71.4%) and specific (87.8%) when using these cutoffs. Using these cutoffs for 41 patients with negative DAT referred to our laboratory in 2006, all the pseudonegative cases were treated with steroids before the test. The 31 untreated cases could be grouped using one cutoff value of 78.5 and showed 100% sensitivity and 94% specificity, independent of gender and age. Results indicate that RBC‐IgG could become a standard approach for the diagnosis of Coombs‐negative AIHA, when measured before treatment. Am. J. Hematol., 2009.

Characterization of direct antiglobulin test-negative autoimmune hemolytic anemia: A study of 154 cases

Direct antiglobulin test (DAT)‐negative (DAT‐)autoimmune hemolytic anemia (AIHA) is empirically thought to show the same clinical conditions as DAT‐positive (DAT+)AIHA, with the exception of an adequate amount of red blood cell (RBC)‐bound immunoglobulin (Ig)G. We investigated the clinical characteristics of DAT−AIHA in comparison with DAT+AIHA. Of the 582 patients referred to our laboratory with undiagnosed hemolytic anemia, AIHA was clinically diagnosed in 216 patients (DAT−AIHA, n = 154; DAT+AIHA, n = 62). The percentage of reticulocytes, mean corpuscular volume, RBC‐IgG levels, white blood cell count, and total protein (TP) levels were significantly higher in patients with DAT+AIHA than patients with DAT−AIHA. The hemoglobin level was significantly lower in patients with DAT+AIHA. No significant differences between patients with DAT−AIHA and DAT+AIHA existed with respect to age, gender, idiopathic/secondary nature, complications such as Evans syndrome, effectiveness of steroid treatment, or survival rate at 1 year following diagnosis. Patients with DAT−AIHA required significantly lower doses of steroids for maintenance therapy. Based on multivariate analysis of idiopathic DAT−AIHA (n = 110), TP and Evans syndrome were associated with the effectiveness of steroids (adjusted odds ratio [aOR], 1.36/[0.1 g/dl]; 95% confidence interval [CI], 1.01–1.84) and survival at the 1‐year follow‐up (aOR, 0.1; 95% CI, 0.01–0.88). Our results indicate that patients with DAT−AIHA generally suffer milder anemia and hemolysis than patients with DAT+AIHA, respond equally well to steroids, and have comparable survival at 1‐year. Am. J. Hematol. 88:93–96, 2013.

Aly/REF, a factor for mRNA transport, activates RH gene promoter function

The rhesus (Rh) blood group antigens are of considerable importance in transfusion medicine as well as in newborn or autoimmune hemolytic diseases due to their high antigenicity. We identified a major DNaseI hypersensitive site at the 5′ flanking regions of both RHD and RHCE exon 1. A 34 bp fragment located at −191 to −158 from a translation start position, and containing the TCCCCTCCC sequence, was involved in enhancing promoter activity, which was assessed by luciferase reporter gene assay. A biotin‐labelled 34 bp probe isolated an mRNA transporter protein, Aly/REF. The specific binding of Aly/REF to RH promoter in erythroid was confirmed by chromatin immunoprecipitation assay. The silencing of Aly/REF by siRNA reduced not only the RH promoter activity of the reporter gene but also transcription from the native genome. These facts provide second proof of Aly/REF as a transcription coactivator, initially identified as a coactivator for the TCRα enhancer function. Aly/REF might be a novel transcription cofactor for erythroid‐specific genes.

Molecular characterization of weak D phenotypes by site-directed mutagenesis and expression of mutant Rh–green fluorescence protein fusions in K562 cells

Mutations detected in 161 weak D samples from Caucasians have been classified into 16 types. Because flow cytometry using monoclonal anti‐D antibodies (mAbs) has shown that weak D red cells display type‐specific antigen density, these mutations in transmembranous regions have been assigned weak D phenotypes. The present study attempts to confirm or refute this assignment.

A new mutation detected in RhAG of a Japanese family with Rhmod syndrome may form a longer RhAG protein

We have read with interest the article on bacterial contamination of whole blood by de Korte et al.1 In the study, the authors found bacterial contamination in 0.34 percent of the whole-blood units tested. Others have reported a wide range of bacterial contamination rates for different blood components.2-4 We are concerned that the differences may depend more on test methods than on the type of blood component analyzed. For whole blood, the percentage of positive results ranges from 0.34 percent, as reported by de Korte et al., to 2.2 percent, as reported in the study of Bruneau et al.2 In a recent review on bacterial contamination in platelet concentrates, the percentage of positive results ranged from 0.08 to 0.8 percent.3,4 We conducted a prospective study by culturing 9232 units of random-donor platelet concentrates using an automated microbe detection system (Bact/Alert, Organon Teknika, Boxtel, The Netherlands).5 Similar to Goldman and Blajchman,3 we focused on careful donor arm disinfection and aseptic sampling, and we used a confirmation algorithm (Table 1). All samples that were reactive in the Bact/Alert were confirmed by routine culture. Samples that did not react were considered negative for bacteria. Each reactive sample with bacteria growth on the routine culture was subcultured for identification of the bacteria, and a second sample from the same blood component was cultured (retest). A reactive result with the detection system (Bact/Alert) was interpreted in three different ways. First, the result could be a false positive, reported when the subculture gave a negative result. This was usually caused by machine failure. The second possibility, an unconfirmed positive result, was reported when the subculture showed bacterial growth and the species was identified but the retest was negative or a different bacteria species was identified. An unconfirmed positive result was likely to be due to contamination during sampling. The third possibility, a confirmed positive result, was reported when the subculture showed bacterial growth and the bacterial species was identified, with the retest showing growth and identical species identification. In our study, we found a confirmed positive result rate of 0.03 percent, similar to that of Goldman and Blajchman.3 All together, our rate of initially reactive samples (Bact/Alert) was between 0.4 and 0.6 percent. The confirmed positive result rate was five times greater when 5 units of random-donor platelets were cultured in the same culture bottle (pool testing). The rate was 1.4 percent during the first 12 weeks of the study, which showed a characteristic learning curve for the technologists who performed the sampling. We believe that the higher rates observed with pooling and during the beginning of our study were due to contamination of the samples during the study. De Korte et al.1 reported careful arm disinfection and sampling methods, but the method used to identify contamination caused by sampling was not well defined. Thus, the rate of true positive results could be lower than the rate reported. We think that because contamination during sample manipulation is not completely avoidable, a confirmatory algorithm should be used. We understand that samples that are truly positive could test negative in the retest, but the use of a confirmatory algorithm would allow reporting of a more complete picture of the true prevalence of bacterial contamination of blood components and permit a better comparison among the results of different studies. Emma Castro, MD e-mail: ecastro@cdscruzroja.infonegocio.com José L. Bueno, MD Centro de Donación de Sangre Cruz Roja Española C/Juan Montalvo 3, bajo 28040 Madrid, Spain

Entire sequence of a mouse chromosomal segment containing the gene Rhced and a comparative analysis of the homologous human sequence.

The mouse genomic sequence of the region containing the gene Rhced, the orthologue to the human gene RH30, was determined to elucidate the structure of Rhced and its flanking regions and to compare these with the corresponding human genomic region. Two genes, Smp1 and AK003528 (an orthologue of FLJ10747), flank Rhced. Neither sequences homologous to the characteristic nucleotide elements flanking the RHD gene in humans (rhesus boxes) nor an additional Rh gene were found within the mouse region sequenced. This result and that of a previous report demonstrate that this chromosomal region of the mouse comprises five genes (FLJ10747-RHCE-SMP1-NPD014-P29) that exhibit syntenic homology with the corresponding human region, which suggests that the RHD gene and rhesus boxes were inserted later. Evaluations of tissue distribution and subcellular localization of these genes indicate that the SMP1 orthologue has a ubiquitous tissue distribution and cytoplasmic localization, whereas AK003528 is expressed slightly higher in testis with a strong subcellular localization in the nucleus. Despite the steady improvements in the draft sequence of the human genome, this study demonstrates the continuing benefits of comparative genetic analyses in increasing our understanding of human genomic structure.

Carotid plaque is a new risk factor for peripheral vestibular disorder: a retrospective cohort study

Abstract Many chronic diseases are associated with dizziness or vertigo, as is peripheral vestibular disorder (PVD). Although carotid plaque development is linked to atherosclerosis, it is unclear whether such plaques can lead to the development of PVD. We therefore conducted this study to investigate the presence of an association between carotid plaque and new PVD events. In this retrospective study, we consecutively enrolled 393 patients ≥20 years old who had been treated for chronic diseases such as hypertension, dyslipidemia, and diabetes mellitus for ≥6 months at a primary care clinic (Oki Clinic, Japan) between November 2011 and March 2013. Carotid plaque presence was measured with high-resolution ultrasonography for all patients. During a 1-year follow-up period, an otorhinolaryngologist diagnosed and reported any new PVD events (the main end point). Hazard ratios (HRs) and 95% confidence intervals (CIs) for new PVD occurrence were estimated using the Cox proportional hazard regression model. The mean age of the participants was 65.5 years; 33.8% were men, and 12.7%, 82.4%, and 93.1% had diabetes mellitus, hypertension, and dyslipidemia, respectively. There were 76 new PVD events; patients with carotid plaque had a greater risk of such events (crude HR: 3.25; 95% CI: 1.62–6.52) compared to those without carotid plaque. This risk was even higher after adjusting for traditional risk factors for atherosclerosis (adjusted HR: 4.41; 95% CI: 1.75–11.14). Carotid plaques are associated with an increased risk of new PVD events.

Incidence of dizziness and vertigo in Japanese primary care clinic patients with lifestyle-related diseases: an observational study

Objective Dizziness and vertigo are highly prevalent symptoms among patients presenting at primary care clinics, and peripheral vestibular disorder (PVD) is their most frequent cause. However, the incidence of PVD has not been well documented. This study aimed to investigate the incidence of dizziness, vertigo, and PVD among patients presenting at a primary care clinic. Design This was an observational study. Setting and participants Between November 2011 and March 2013, we observed 393 patients, all at least 20 years old, who had been treated for chronic diseases such as hypertension, dyslipidemia, and diabetes mellitus for at least 6 months at a primary clinic (Oki Clinic) in Japan. Outcome The main outcome of interest was new incidence of dizziness, vertigo, and PVD events. During the 1-year follow-up period, the otorhinolaryngologist diagnosed and reported new PVD events. Results The mean age of the 393 participants at entry was 65.5 years. Of the study participants, 12.7%, 82.4%, and 92.6% had diabetes mellitus, hypertension, and dyslipidemia, respectively. We followed up all the participants (100%). During the 662.5 person-years of follow-up, 121 cases of dizziness or vertigo (dizziness/vertigo) and 76 cases of PVD were observed. The incidence of dizziness/vertigo and PVD was 194.7 (95% confidence interval: 161.6–232.6) per 1,000 person-years and 115.7 (95% confidence interval: 92.2–142.6) per 1,000 person-years, respectively. There were 61 cases of acute peripheral vestibulopathy, 12 of benign paroxysmal positional vertigo, and three of Meniere’s disease among the 76 PVD patients. Conclusion We reported the incidence of dizziness/vertigo among Japanese primary care clinic patients, which was higher than that usually observed in the general population. Furthermore, we described the incidence of PVD and found that it was a major cause of dizziness/vertigo.

Coombs Negative Autoimmune Hemolytic Anemia in Crohn’s Disease

Patient: Female, 41 Final Diagnosis: Coombs negative autoimmune hemolytic anemia Symptoms: Dark urine • dizziness • dyspnea Medication: — Clinical Procedure: Immunoradiometric assay for RBC-IgG Specialty: Hematology Objective: Rare disease Background: Anemia is a common, important extraintestinal complication of Crohn’s disease. The main types of anemia in patients with Crohn’s disease are iron deficiency anemia and anemia of chronic disease. Although patients with Crohn’s disease may experience various type of anemia, autoimmune hemolytic anemia (AIHA) in patients with Crohn’s disease, especially Coombs-negative AIHA, is very rare. Case Report: A 41-year-old woman with Crohn’s disease presented to our emergency room (ER) with dark urine, dizziness, and shortness of breath. The activity of Crohn’s disease had been controlled, with Crohn’s disease activity index (CDAI) score below 100 point. On physical examination, the patient had pale conjunctivae and mildly icteric sclerae. Serum bilirubin was raised at 3.1 mg/dL, lactate dehydrogenase (LDH) level was 1418 U/L and the haptoglobin level was <3 mg/dL. Results of direct and the indirect Coombs tests were all negative. We then measured the RBC-IgG to evaluate the possibility of Coombs-negative AIHA. The result revealed that RBC-IgG level was 352 IgG molecules/cell, with the cut-off value at 78.5 IgG molecules/cell. Conclusions: We report a case of Coombs-negative AIHA in a patient with Crohn’s disease with chronic anemia, diagnosed by red blood cell-bound immunoglobulin G (RBC-IgG) and treated with steroids therapy.