Toru Nabika

A novel polymorphism of the brain-derived neurotrophic factor (BDNF) gene associated with late-onset Alzheimer's disease

Several lines of evidence have suggested altered functions of the brain-derived neurotrophic factor (BDNF) in the pathogenesis of neurodegenerative diseases including Alzheimers disease (AD). In the search for polymorphisms in the 5′-flanking and 5′-noncoding regions of the BDNF gene, we found a novel nucleotide substitution (C270T) in the noncoding region. We performed an association study between this polymorphism and AD in a Japanese sample of 170 patients with sporadic AD (51 early-onset and 119 late-onset) and 498 controls. The frequency of individuals who carried the mutated type (T270) was significantly more common in patients with late-onset AD than in controls (Pu2009=u20090.00004, odds ratio: 3.8, 95% CI 1.9–7.4). However, there was no significant difference in the genotype distribution between the patients with early-onset AD and the controls, although this might be due to the small sample size of the early-onset group. Our results suggest that the C270T polymorphism of the BDNF gene or other unknown polymorphisms, which are in linkage disequilibrium, give susceptibility to late-onset AD. We obtained no evidence for the possible interactions between the BDNF and apolipoprotein E (APOE) genes, suggesting that the possible effect of the BDNF gene on the development of late-onset AD might be independent of the APOE genotype.

Lack of Evidence for Association Between the Endothelial Nitric Oxide Synthase Gene and Hypertension

Significant association between a Glu298Asp polymorphism of the endothelial nitric oxide synthase (eNOS) gene and essential hypertension was recently reported in Japanese populations, with the 298Asp variant showing a higher prevalence in hypertensive patients (10.3% to 12.0%) than in normotensive subjects (5.0% to 5.8%). In contrast, another study demonstrated that the 298Glu variant was significantly associated with hypertension in a Caucasian population. We therefore undertook an extensive association study in Japanese to resolve these contradictory claims. A total of 1165 individuals were selected from clinic outpatients and hospital staff in a single institution. The relevance of the Glu298Asp polymorphism to hypertension in this population was tested in 2 ways. First, a case-control study was conducted in 549 hypertensive and 513 normotensive subjects within the study population, with the chi2 statistic used to test the significance of an association between eNOS genotype and the presence of hypertension. Second, an ANOVA was used to test the significance of an association between eNOS genotype and the level of blood pressure within the entire population except for 167 hypertensive subjects who had been under treatment for hypertension. No significant association was observed in either of the statistics tested. Allele frequencies of 298Asp were concordant across the panels: 8.4% in hypertensive subjects, 8. 2% in normotensive subjects, and 7.9% and 9.5% in 2 additional sample populations used as reference panels. Taken together, our results do not support the previous observation that the molecular variant of the eNOS gene may confer principal susceptibility for essential hypertension but rather suggest the existence of sampling variation.

Evaluation of Genetic Risk Factors for Silent Brain Infarction

BACKGROUND AND PURPOSEnSilent brain infarction (SBI) is often found with white matter hyperintensity. A recent genetic study on elderly twins indicated that the susceptibility to white matter hyperintensity was largely determined by genetic factors, implying the existence of genetic susceptibility for SBI as well. We therefore studied 3 genetic polymorphisms in SBI, the deletion/insertion polymorphism of angiotensin-converting enzyme (ACE) gene, the apolipoprotein(a) [apo(a)] size polymorphism, and the T677C polymorphism of methylenetetrahydrofolate reductase (MTHFR) gene, by a case-control study.nnnMETHODSnBy MRI, 147 subjects with SBI and 214 without cerebral infarctions (control group) were selected from participants of a health examination of the brain. Seventy-four patients with symptomatic subcortical infarction (SSI) from the same area were also included in the study. In addition to the control group, 2 more reference populations were recruited. Typing of the apo(a) size polymorphism was done by Western blotting with the use of an anti-apo(a) antibody. Genotypes of ACE and MTHFR were determined by polymerase chain reaction amplification of the genomic DNA and subsequent restriction enzyme digestion.nnnRESULTSnThe ACE polymorphism was not associated with either SBI or SSI. In contrast, the small apo(a) was associated with both SSI and SBI. The MTHFR polymorphism was associated only with SSI. The association of MTHFR and apo(a) was greater in the younger subjects.nnnCONCLUSIONSnAmong the 3 genetic polymorphisms studied, only the apo(a) size polymorphism is a risk factor for SBI.

Silent stroke: pathogenesis, genetic factors and clinical implications as a risk factor.

Silent stroke is frequently recognized in elderly persons. Diffusion-weighted magnetic resonance imaging has proved to be highly sensitive in the detection of recent silent stroke, and may further applications in the future. Silent stroke in healthy and asymptomatic individuals mainly comprises lacunar infarcts, which are often associated with white matter changes. Thus, analyses of risk factors and genetic factors in small-vessel diseases such as lacunar infarct and white matter changes may provide clues regarding the pathogenesis of silent stroke. Silent stroke may be a risk factor for clinical stroke and cognitive impairment, but prospective studies are required to confirm this.

Aging and salt-loading modulate blood pressure QTLs in rats☆

To evaluate the effects of nongenetic factors, aging, and salt-loading on the quantitative trait loci (QTLs) for blood pressure (BP), we conducted a genome-wide linkage analysis using multiple sets of BP measurements in 125 male F2 generation cross derived from stroke-prone spontaneously hypertensive rats and normotensive Wistar-Kyoto rats. The experiment was arranged in two stages. In the first stage, corresponding to the developing period of the rats, BP was measured repeatedly without loading of salt; this continued until the rats were 5 months of age. In the second stage, after the baseline BP leveled off, 1% salt water was given to the rats and BP was monitored for the subsequent 7 months. Genome scanning was performed using 201 markers. In the developing period, three QTLs were identified on chromosomes 1, 3, and 4 (logarithmic odds [LOD] scores of 5.6, 3.1, and 3.2, respectively), which had peaks at 8 or 10 weeks of age. In the latter salt-loading stage, QTLs for BP were detected on chromosomes 1 and 10 (LOD scores 4.6 and 4.5, respectively). When the BP increase during salt-loading was analyzed as a phenotype, however, only the region on chromosome 10 showed linkage at a suggestive level (LOD score 3.2). The present study provides experimental evidence that QTLs for BP could be modulated by nongenetic factors, such as aging and salt-loading.

Alzheimer's disease and 5-HTTLPR polymorphism of the serotonin transporter gene: no evidence for an association.

Recently two independent research groups consistently reported a significant association between the serotonin transporter (5-HTT) gene and late-onset sporadic Alzheimers disease (AD). They found that the short allele of the 5-HTT gene-linked polymorphic region (5-HTTLPR), which is associated with reduced transcriptional activity of the gene, increases the risk of developing late-onset AD. The present study tried to replicate this finding in a Japanese sample. We genotyped 41 patients with early-onset AD (<65 years), 82 with late-onset AD, and 336 controls. There was no significant difference in genotype or allele distribution between either patient group and controls in our sample, suggesting that the 5-HTTLPR does not play a major role in the pathogenesis of AD in Japanese.

Identification of Quantitative Trait Loci for Serum Cholesterol Levels in Stroke-Prone Spontaneously Hypertensive Rats

The stroke-prone spontaneously hypertensive rat (SHRSP) has been reported to show significantly lower levels of serum total cholesterol than the normotensive control strain Wistar-Kyoto rat (WKY). Because selective inbreeding was conducted for stroke proneness, this concomitantly inherited characteristic of SHRSP may play some pathophysiological role in stroke. We evaluated the genetic determinants of the cholesterol trait by estimating heritability and subsequently by undertaking a genome-wide screen with 161 genetic markers in F(2) progeny involving SHRSP and WKY (104 male and 106 female rats). Three quantitative trait loci (QTLs) were detected on rat chromosomes 5, 7, and 15. Markers from the linked region on chromosome 15 indicated significant evidence of linkage with a maximal log of the odds (LOD) score of 7.7, whereas those on chromosomes 5 and 7 cosegregated with the trait in a sex-specific manner (the QTL close to genetic marker D5 Mit5 reached an LOD score of 7.3 in males, and that close to D7 Mit10 reached an LOD score of 3.2 in females). The male-specific QTL on chromosome 5 appeared to overlap with previously reported QTLs for stroke-associated phenotypes, but an identical gene (or genes) appeared unlikely to control these and the cholesterol traits simultaneously. In the present study, serum cholesterol levels were shown to be highly genetically determined in SHRSP (the heritability estimates are 76% in males and 83% in females), and 3 QTLs with substantial effects were identified. Further work, however, is required to clarify whether the cholesterol trait is related to the etiology of stroke or has been retained by chance through the inbreeding process in SHRSP.

Congenic Rats For Hypertension: How Useful Are They For The Hunting Of Hypertension Genes?

1. Linkage studies have revealed quantitative trait loci (QTL) for blood pressure in the rat genome using genetic hypertensive rat models. To identify the genes responsible for hypertension, the construction of congenic rats is essential.

Evaluation of the poly(ADP-ribose) polymerase gene in human stroke

Nitric oxide (NO) and its reactant product, peroxynitrite, have been implied to mediate neuronal damage following cerebral ischemia. However, the cellular targets of these compounds remain unclear. Studies using poly(ADP-ribose) polymerase (PARP) inhibitors and PARP knock-out mice have recently demonstrated that excessive activation of this nuclear enzyme plays an important role in NO-induced neurotoxicity. To evaluate the relevance of this plausible candidate gene to human stroke, we undertook a case-control study in Japanese. Participants comprised 213 cerebral infarction cases and 374 age- and sex-matched controls. As a primary investigation, we screened polymorphic sites of the PARP gene, and newly identified a total of four polymorphisms in 1230-bp 5-flanking sequence. None of them were, however, located on the known promoter components of the gene. Two bi-allelic polymorphisms selected and a CA-repeat polymorphism were subsequently characterized in the case-control study, but none were significantly associated with cerebral infarction in the present study. Our data thus suggest that the tested PARP polymorphisms do not principally contribute to cerebral infarction, although extensive searches would be required to clarify whether the PARP gene plays an important role in the pathogenesis of human stroke.

Strain differences in SA gene expression in brain and kidney of normotensive and hypertensive rats.

AbstractSUMMARYn 1. In situ hybridization done using a 35S-cRNA probe was carried out to obtain information on the expressions of the SA gene in brains and kidneys of the spontaneously hypertensive rat (SHR) strain obtained from the Izumo colony (/Izm) and from Charles River Laboratories (/Crj).2. In the brain, SA mRNA expression was most abundantly observed in epithelial cells of the choroid plexus. High to moderate levels was present on neurons of the CA1–CA4 pyramidal cell layer and the dentate gyrus of the hippocampus and the cerebellar Purkinje cell layer. The solitary tract nucleus and the dorsal motor nucleus of the vagus expressed the SA gene at very low levels. An increase in the expression was noted in the choroid plexus of WKY/Crj; there was no difference, however, in expression levels of other brain areas between WKY/Izm, SHR/Izm, and SHRSP/Izm, and between WKY/Crj and SHR/Crj.3. In the kidney, expression signals of SA mRNA were observed in renal medullary rays and focal cortex of WKY/Izm, SHR/Izm, SHRSP/Izm, and SHR/Crj, whereas mRNA expression in the WKY/Crj kidney was observed in medullary rays and outer strips of the outer medulla. Microscopically, hybridization signals were predominant in the proximal tubules.4. Expression densities decreased only in the kidney of WKY/Crj in 4-and 8-week-old rats, but not in the WKY/Izm kidney, compared with findings in SHR and SHRSP kidneys. These observations are in good agreement with data from Northern blot analysis.5. The SA gene expressions in the brain and the kidney seem not to relate to states of elevated blood pressure, but rather to strain differences. Abundant expressions in the brain and the kidney may mean that the SA gene plays a role in the water–electrolyte transport system. It is noteworthy that there are neuronal expressions of the SA gene in hippocampal pyramidal cells and cerebellar Purkinje cells.