CST3 Gene Research Articles

Dominant CST3 variants cause adult onset leukodystrophy without amyloid angiopathy.

Leukodystrophies are rare genetic white matter disorders that have been regarded as mainly occurring in childhood. This perception has been altered in recent years, as a growing number of leukodystrophies have been described as having an onset in adulthood. Still, many adult patients presenting with white matter changes remain without a specific molecular diagnosis. We describe a novel adult onset leukodystrophy in 16 patients from eight families carrying one of four different stop-gain or frameshift dominant variants in the CST3 gene. Clinical and radiological features differ markedly from the previously described Icelandic cerebral amyloid angiopathy found in patients carrying p.Leu68Asn substitution in CST3. The clinical phenotype consists of recurrent episodes of hemiplegic migraine associated with transient unilateral focal deficits and slowly progressing motor symptoms and cognitive decline in mid to older adult ages. In addition, in some cases acute onset clinical deterioration led to a prolonged episode with reduced consciousness and even early death. Radiologically, pathognomonic changes are found at typical predilection sites involving the deep cerebral white matter sparing a periventricular and directly subcortical rim, the middle blade of corpus callosum, posterior limb of the internal capsule, middle cerebellar peduncles, cerebral peduncles and specifically the globus pallidus. Histopathologic characterization in two autopsy cases did not reveal angiopathy, but instead micro- to macrocystic degeneration of the white matter. Astrocytes were activated at early stages and later displayed severe degeneration and loss. In addition, despite the loss of myelin, elevated numbers of partly apoptotic oligodendrocytes were observed. A structural comparison of the variants in CST3 suggests that specific truncations of cystatin C result in an abnormal function, possibly by rendering the protein more prone to aggregation. Future studies are required to confirm the assumed effect on the protein and to determine pathophysiologic downstream events at the cellular level.

Multiomic Analyses of Dopaminergic Neurons Isolated from Human Substantia Nigra in Parkinson’s Disease: A Descriptive and Exploratory Study

Dopaminergic neurons (DA) of the substantia nigra pars compacta (SNpc) selectively and progressively degenerate in Parkinson’s disease (PD). Until now, molecular analyses of DA in PD have been limited to genomic or transcriptomic approaches, whereas, to the best of our knowledge, no proteomic or combined multiomic study examining the protein profile of these neurons is currently available. In this exploratory study, we used laser capture microdissection to extract regions from DA in 10 human SNpc obtained at autopsy in PD patients and control subjects. Extracted RNA and proteins were identified by RNA sequencing and nanoliquid chromatography–mass spectrometry, respectively, and the differential expression between PD and control group was assessed. Qualitative analyses confirmed that the microdissection protocol preserves the integrity of our samples and offers access to specific molecular pathways. This multiomic analysis highlighted differential expression of 52 genes and 33 proteins, including molecules of interest already known to be dysregulated in PD, such as LRP2, PNMT, CXCR4, MAOA and CBLN1 genes, or the Aldehyde dehydrogenase 1 protein. On the other hand, despite the same samples were used for both analyses, correlation between RNA and protein expression was low, as exemplified by the CST3 gene encoding for the cystatin C protein. This is the first exploratory study analyzing both gene and protein expression of laser-dissected neuronal parts from SNpc in PD. Data are available via ProteomeXchange with identifier PXD024748 and via GEO with identifier GSE 169755.

Association of CST3 Gene with Its Protein: Cystatin C in Health and Severe Periodontal Disease.

Background: Cystatin C (CSTC), a cysteine protease inhibitor, is found to be elevated in periodontal disease in an attempt to counterbalance the proteolytic enzymes and increased osteoclastic activity. Evidence on CSTC levels in periodontal health and disease has reported contradicting results, making its role as a biomarker in periodontal pathogenesis inconclusive. Aim: To evaluate CST3 gene expression and correlate it with CSTC levels in periodontal health and severe periodontal disease. Materials and Methods: A total of 50 patients with 25 in each group (Group I-periodontally healthy, Group II-Stage III/IV periodontitis) were recruited. Clinical parameters were assessed following which gingival crevicular fluid and gingival tissue samples were collected from tooth deemed for extraction. CSTC protein level and CST3 gene expression were analyzed using enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction, respectively. Results: Elevated concentrations of CSTC protein and CST3 gene expression were observed in Group II in comparison with Group I, which was considered statistically significant (p < 0.001). Further, a highly significant (p < 0.001) positive correlation was witnessed between CSTC protein and CST3 gene in both groups. In addition, the overall correlation between CSTC protein, CST3 gene, and clinical parameters was positive and highly significant (p < 0.001). Conclusion: CSTC protein levels and CST3 gene expression were significantly higher in periodontal disease compared with health, and there was a positive correlation between the gene and protein levels. Therefore, it can be concluded that CST3 gene can be used as a reliable indicator of periodontal disease pathogenesis. Clinical Trial Registration number: CTRI/2020/03/023926.

Pressure overload promotes cystatin C secretion of cardiomyocytes to regulate the MAPK signaling pathway and mediate cardiac hypertrophy.

BackgroundThis study aimed to compare serum cystatin C (CysC) levels between hypertensive and non-hypertensive patients, and to explore the correlation between serum CysC and left ventricular hypertrophy (LVH). We also investigated the effects of pressure overload on cardiac expression and secretion of CysC, and explored the direct effect of CysC on the hypertrophy of primary cardiomyocytes.MethodsSerum CysC was compared in patients with hypertension (634 patients) and those without hypertension (411 patients), and the correlation between serum CysC levels and LVH was explored. A transverse aortic constriction (TAC) mouse model and a mechanical stretch model of primary cardiomyocytes and fibroblasts were developed to compare cardiac expression and secretion of CysC under pressure overload. After intervention with exogenous CysC, we compared the cross-sectional area of primary cardiomyocytes, cardiac hypertrophy-associated gene expression, and phosphorylation of the MAPK signaling pathway.ResultsIn chronic kidney disease (CKD) stage 1 patients, serum CysC was higher in hypertensive patients independent of renal function. Serum CysC elevation was an independent predictor of LVH after correction for endogenous creatinine clearance rate (eCCr), left ventricular ejection fraction (LVEF), and NT-proBNP. Cardiac levels of CysC in TAC mice were elevated. CST3 gene expression was upregulated, and both intracellular and culture supernatant CysC levels increased after mechanical stretch of primary cardiomyocytes. After intervention with exogenous CysC, the cross-sectional area of primary cardiomyocytes increased, as well as the gene expression of Nppa, Nppb, and Myh7, and the phosphorylation of ERK, p38, and TAK1.ConclusionsSerum CysC levels were higher in hypertensive patients, and serum CysC elevation was an independent predictor of LVH after correction for eCCr. Pressure overload induced greater cardiomyocyte secretion of CysC. Exogenous CysC can enter cardiomyocytes, having a pro-hypertrophic effect on primary cardiomyocytes through regulation of the MAPK signaling pathways.

Generation of Gene-Knockout Mongolian Gerbils via CRISPR/Cas9 System.

The Mongolian gerbil (Meriones unguiculatus), a well-known “multifunctional” experimental animal, plays a crucial role in the research of hearing, cerebrovascular diseases and Helicobacter pylori infection. Although the whole-genome sequencing of Mongolian gerbils has been recently completed, lack of valid gene-editing systems for gerbils largely limited the further usage of Mongolian gerbils in biomedical research. Here, efficient targeted mutagenesis in Mongolian gerbils was successfully conducted by pronuclear injection with Cas9 protein and single-guide RNAs (sgRNAs) targeting Cystatin C (Cst3) or Apolipoprotein A-II (Apoa2). We found that 22 h after human chorionic gonadotropin (hCG) injection, zygote microinjection was conducted, and the injected zygotes were transferred into the pseudopregnant gerbils, which were induced by injecting equine chorionic gonadotropin (eCG) and hCG at a 70 h interval and being caged with ligated male gerbils. We successfully obtained Cst3 and Apoa2 gene knockout gerbils with the knockout efficiencies of 55 and 30.9%, respectively. No off-target effects were detected in all knockout gerbils and the mutations can be germline-transmitted. The absence of CST3 protein was observed in the tissues of homozygous Cst3 knockout (Cst3-KO) gerbils. Interestingly, we found that disruption of the Cst3 gene led to more severe brain damage and neurological deficits after unilateral carotid artery ligation, thereby indicating that the gene modifications happened at both genetic and functional levels. In conclusion, we successfully generated a CRISPR/Cas9 system based genome editing platform for Mongolian gerbils, which provided a foundation for obtaining other genetically modified gerbil models for biomedical research.

Association between cystatin C gene polymorphism and the prevalence of white matter lesion in elderly healthy subjects

Cystatin C (CST3) is a cysteine protease inhibitor abundant in the central nervous system, and demonstrated to have roles in several pathophysiological processes including vascular remodeling and inflammation. Previously, we showed a relation of CST3 gene polymorphisms with deep and subcortical white matter hyperintensity (DSWMH) in a small case-control study. In this study, we aimed to investigate the relation in a larger cross-sectional study. Participants of a brain health examination program were recruited (n = 1795) in the study, who underwent routine blood tests and cognitive function tests. Cerebral white matter changes were analyzed by MRI. Additionally, 7 single nucleotide polymorphisms (SNPs) (−82G/C, −78T/G, −5G/A, +4A/C, +87C/T, +148G/A and +213G/A) in the promoter and coding regions of CST3 gene were examined. Among them, carriers of the minor allele haplotype −82C/+4C/+148A were significantly associated with decreased CST3 concentration in the plasma. Unadjusted analysis did not show significant relation between carriers of the minor allele haplotype and periventricular hyperintensity (PVH), but DSWMH was marginally (p < 0.054) increased in this group. After adjusting the effects of other variables like age and kidney function, logistic regression analysis revealed that carriers of the minor allele haplotype were at a significantly increased risk of developing both PVH and DSWMH. Thus, our results suggest that carriers of the minor allele haplotype −82C/+4C/+148A of CST3 gene could be at an increased risk to develop cerebral white matter disturbance.

Effects of CST3 Gene G73A Polymorphism on Cystatin C in a Prospective Multiethnic Cohort Study

Background/Aims: G73A polymorphism in the CST3 gene of cystatin C has been associated with Alzheimer’s disease, age-related macular degeneration, and cardiovascular disease. However, studies investigating the influence of this genetic variability on serum cystatin C and cystatin-based renal function estimate are limited. Therefore, the aim of this study is to investigate the possible association of single-nucleotide polymorphism (rs1064039) of the CST3 gene on the serum cystatin C level and cystatin C-based estimated glomerular filtration rate (eGFR). Methods: Study subjects include patients with various levels of renal function recruited from the nephrology clinic and wards of a tertiary hospital. The blood samples collected were analyzed for serum cystatin C and creatinine levels by particle-enhanced turbidimetric immunoassay and kinetic alkaline picrate method, respectively. DNA was extracted using a commercially available kit. ­Polymerase chain reaction results were confirmed by direct DNA Sanger sequencing. Results: The genotype percentage (G/G = 73%, G/A = 24.1%, and A/A = 2.9%) adhere to the Hardy-Weinberg equilibrium. The dominant allele found in our population was CST3 73G allele (85%). The regression lines’ slope of serum cystatin C against creatinine and cystatin C-based eGFR against creatinine-based eGFR, between G and A allele groups, showed a statistically significant difference (z-score = 3.457, p < 0.001 and z-score = 2.158, p = 0.015, respectively). Patients with A allele had a lower serum cystatin C level when the values were extrapolated at a fixed serum creatinine value, suggesting the influence of genetic factor. Conclusion: Presence of CST3 gene G73A polymorphism affects serum cystatin C levels.

Correlations of serum cystatin C level and gene polymorphism with vascular cognitive impairment after acute cerebral infarction.

The aim of this study was to explore the possible correlations of serum cystatin C level and cystatin C gene (CST3) polymorphism with vascular cognitive impairment in patients who had acute cerebral infarction. A total of 152 patients with acute cerebral infarction were recruited in this case-control study. Patients were divided into vascular cognitive impairment (VCI) group (n = 71) and cognitive impairment no dementia (CIND) group (n = 81). The serum concentrations of cystatin C were measured with immunoturbidimetric assay while the gene polymorphisms of CST3 were determined by technique polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). In the VCI group, serum cystatin C level was significantly higher than that in the control group. The frequency of the B allele was found to be higher in the VCI group as compared with that of the CIND group (18.5% vs 7.7%, p = 0.006). In logistic regression analysis, significant associations of VCI with high serum cystatin C level (OR 3.837 (1.176-12.520), p = 0.026) and CST3 B allele (OR 2.038 (1.048-3.963), p = 0.036) were also found. A high cystatin C level and CST3 B allele confer risks for VCI after acute cerebral infarction. It is probable that measurement of the serum cystatin C level and detection of CST3 gene polymorphism would aid in the early diagnosis of VCI, but further studies are warranted.

Endometrial transcript profile of progesterone-regulated genes during early pregnancy of Water Buffalo (Bubalus bubalis).

Progesterone (P4 ) plays a key role in the establishment and maintenance of pregnancy in most mammals. Unravelling the expression of progesterone-regulated genes can expand the understanding of the embryonic mortality. Accordingly, we studied the relative mRNA expression of the P4 -regulated genes in the buffalo. Uteri were collected from the abattoir and categorized into nonpregnant late luteal phase, stage I (28-38th days of gestation) and stage II (48-56th days of gestation) of pregnancy (n=6/group). After extraction of total RNA from the endometrial tissues, we carried out qRT-PCR for determining the relative mRNA expression of the P4 -regulated genes using nonpregnant late luteal phase as calibrator group. The expression of LGALS3BP (essential for maternal recognition of pregnancy) gene was found to be significantly upregulated (p<0.05), while MUC1 (important for embryo attachment) gene was downregulated in stage I and II of pregnancy. We observed no significant change in the expression of LGALS1, LGALS9 and CTSL genes. The SLC5A11 and SLC2A1 genes (involved in the transport of glucose to endometrium) in early pregnancy were upregulated in the pregnancy stage I (p<0.05) relative to nonpregnant late luteal phase. The CST3 gene was significantly upregulated in pregnancy stage II (p<0.01). These results provide molecular insights into the specific pathways involved in foeto-maternal communication during early pregnancy in buffaloes.

Genome-Wide Association Studies in Nephrology: Using Known Associations for Data Checks

Prior to conducting genome-wide association studies (GWAS) of renal traits and diseases, systematic checks to ensure data integrity and analytical work flow should be conducted. Using positive controls (ie, known associations between a single-nucleotide polymorphism [SNP] and a corresponding trait) allows for identifying errors that are not apparent solely from global evaluation of summary statistics. Strong genetic control associations of chronic kidney disease (CKD), as derived from GWAS, are lacking in the non-African ancestry CKD population; thus, in this perspective, we provide examples of and considerations for using positive controls among patients with CKD. Using data from individuals with CKD who participated in the CRIC (Chronic Renal Insufficiency Cohort) Study or PediGFR (Pediatric Investigation for Genetic Factors Linked to Renal Progression) Consortium, we evaluated 2 kinds of positive control traits: traits unrelated to kidney function (bilirubin level and body height) and those related to kidney function (cystatin C and urate levels). For the former, the proportion of variance in the control trait that is explained by the control SNP is the main determinant of the strength of the observable association, irrespective of adjustment for kidney function. For the latter, adjustment for kidney function can be effective in uncovering known associations among patients with CKD. For instance, in 1,092 participants in the PediGFR Consortium, the P value for the association of cystatin C concentrations and rs911119 in the CST3 gene decreased from 2.7×10(-3) to 2.4×10(-8) upon adjustment for serum creatinine-based estimated glomerular filtration rate. In this perspective, we give recommendations for the appropriate selection of control traits and SNPs that can be used for data checks prior to conducting GWAS among patients with CKD.

&lt;i&gt;In silico&lt;/i&gt; prediction of functional loss of cst3 gene in hereditary cerebral amyloid angiopathy

The computational identification of missense mutation in CST3 (CYSTATIN 3 or CYSTATIN C) gene has been done in the present study. The missense mutations in the CST3 gene will leads to hereditary cerebral amyloid angiopathy The initiation of the analysis was done with SIFT followed by POLYPHEN-2 and I-Mutant 2.0 using 24 variants of CST3 gene of Homo sapiens which were derived from dbSNP. The analysis showed that 5 variants (Y60C, C123Y, L19P, Y88C, L94Q) were found to be less stable and damaging by SIFT, POLYPHEN-2 and I-MUTANT2.0. Furthermore the outputs of SNP & GO are collaborated with PHD-SNP (Predictor of Human Deleterious-Single Nucleotide Polymorphism) and PANTHER to predict 5 variants (Y60C, Y88C, C123Y, L19P, and L94Q) having clinical impact in causing the disease. These findings will be certainly helpful for the present medical practitioners for the treatment of cerebral amyloid angiopathy. DOI: http://dx.doi.org/10.3329/bjp.v8i4.16524 Bangladesh Journal of Pharmacology Vol.8(4) 2013 390-394

Contribution of Cystatin C Gene Polymorphisms to Cerebral White Matter Lesions

Background: Vascular remodeling plays an important role in the development of arteriosclerosis and any of the resulting white matter lesions in the brain. An imbalance between cysteine proteases and the cysteine protease inhibitor cystatin C (CST3) may exacerbate vascular remodeling through degradation of extracellular matrix proteins. Therefore, we evaluated the association between functional polymorphisms in the CST3 gene and the development of cerebral white matter lesions. Methods: In a total of 2,676 participants, 3 CST3 genepolymorphisms were genotyped in 92 cases with severe deep white matter hyperintensity (DWMH), and 184 subjects were randomly selected age- and sex-matched controls without any signs of DWMH. The genetic effects of these polymorphisms on DWMH and plasma CST3 levels were examined. CST3 expression vectors were transfected into an astrocytoma cell line and the expression level of CST3 mRNA was analyzed by quantitative RT-PCR. Intracellular and secreted levels of CST3 in the cell culture were quantified by Western blot and ELISA, respectively. Results: A significant association was found between one CST3 gene haplotype and DWMH (p = 0.002). This haplotype was also associated with lower plasma CST3 levels (p = 0.01). An in vitro transfection study revealed that the +148A allele, which is included in the risk haplotype, significantly reduced the secretion and increased the intracellular accumulation of CST3; however, it had no effect on the mRNA expression. Conclusions: Our study shows that polymorphisms in the CST3 gene are significantly associated with the likelihood of DWMH. Substitution of A for G at +148 of the CST3 gene decreased the extracellular availability of CST3 in vitro, which might result in the activation of protease activity.

Association between CST3 rs2424577 Polymorphism and Corpulence Related Phenotypes during Lifetime in Populations of European Ancestry

Objective: Cystatin C, a protein coded by CST3 gene, is implicated in adipose tissue biology. Our hypothesis is that common variants in CST3 gene could play a role in the development of corpulence during lifetime. Methods: Two tag SNPs were selected to capture all SNPs in the CST3 region. We first investigated the association of the two tag SNPs individually and combined into haplotypes with corpulence related phenotypes in 4,288 French subjects (BMI = 24.31 ( 3.74 kg/m²). Significant findings were replicated in five independent populations – 790 Danish lean men (BMI = 24.63 ( 2.30 kg/m²), 672 Danish obese men (BMI = 33.23 ( 2.34 kg/m²), 763 Swedish women (BMI = 21.73 ( 2.87 kg/m²), 1,848 Danish lean women (BMI = 22.66 ( 2.85 kg/m²) and 2,061 Danish obese women (BMI = 37.01 ( 3.59 kg/m²). Results: Rs2424577 was associated with BMI in three independent populations – G/G carriers were less corpulent than A/A carriers in the French individuals (p = 0.045) and in the Danish lean men (p = 0.021), and they were more corpulent in the group of Swedish women (p = 0.004). This phenomenon has been described as a flip-flop phenomenon, probably caused by a multilocus effect. Conclusion: CST3 rs2424577 is associated with BMI in a complex fashion. This association is probably caused by the interaction between several functional variants.

Quantification of cystatin C in cerebrospinal fluid from various neurological disorders and correlation with G73A polymorphism in CST3

Cystatin C (CC) is a cysteine protease inhibitor abundantly expressed in the central nervous system. Bunina bodies, small eosinophilic intraneuronal inclusions, are stain positive for CC and are the most specific histological hallmark of amyotrophic lateral sclerosis (ALS). In this study, employing a latex turbidimetric immunoassay, levels of CC in cerebrospinal fluid (CSF) were quantified in 130 age-matched individuals with either a neurological disorder [ALS, Alzheimer's disease (AD), Parkinson's disease (PD), tauopathy (TP), multiple system atrophy (MSA), chronic inflammatory demyelinating polyneuropathy (CIDP)] or no known neurological condition (normal control, NC). The CC level in CSF was found to be correlated with the age during the investigation but not the protein concentration. There was no difference in CC levels between NC and ALS or CIDP cases, whereas CC levels were significantly lower in MSA compared with NC. Of the 130 cases, 96 were genotyped, and G/A or A/A polymorphism at + 73 within the CST3 gene was found in 28 individuals. The CC level was significantly lower in the combined group of G/A and A/A genotypes compared with G/G. The present data demonstrate that the level of CC in CSF should not be considered as a biomarker of ALS, but there is a correlation between CC levels and the CST3 genotype.

Flanking Markers of Cystatin C (CST3) Gene Do Not Show Association with Alzheimer’s Disease

Background: In the present study we determined whether the cystatin c gene (CST3) is genetically associated with late-onset Alzheimer’s disease (AD). Methods: Two informative flanking single nucleotide polymorphisms (SNP), rs2424577 and rs3827143, of the CST3 gene and apolipoprotein E (APOE) gene were assessed in 568 Finnish AD patients and 688 cognitively healthy controls. Samples were genotyped with the TaqMan technique, and we conducted a single allele and genotypic distribution comparison as well as an estimation of haplotype frequencies between cases and controls. Results: The APOE genotype distribution differed as expected between the AD cases and controls (p < 0.001). On the whole, any significant differences in AD risk were not found in single SNP and haplotype analyses for the CST3 gene between the whole study cohorts or in the stratified subgroups. Interestingly, AG-genotype carriers of rs3827143 showed a significant difference (p = 0.04) between cases and controls when compared to AA-genotype carriers, but this finding remained insignificant in the adjusted model. Conclusion: Although flanking SNP cover the whole gene transcript with strong linkage disequilibrium, our data show that the CST3 gene is not associated with AD risk in the Finnish population.

Assessment Of The Polymorphic Sacii And Ddei Sites In The 5\' Flanking Region Of The Human Cystatin C Gene In Egyptian Patients

Cystatin C is a cysteine proteinase inhibitor with widespread distribution in body fluids and tissues. Physiologically relevant polymorphisms have been identified in the promoter region and the signal peptide of the cystatin C gene. However, the final importance of cystatin C polymorphisms in human diseases is still discussed controversially and the prognostic value in different conditions is unknown. In this study, in order to assess the significance of the polymorphic genotypes at positions -82, -5 and +4 of the 5' flanking region of the CST3 gene in case of heart, kidney and/or diabetic disorders in Egyptian patients, we compared frequencies of these polymorphisms in various groups of Egyptian patients with different conditions and controls. Genotyping analysis of all subjects revealed that these polymorphisms are in strong-linkage disequilibrium. Notably, no statistically significant differences in the allele or haplotype frequencies were observed for the investigated polymorphisms between patient and control groups. Moreover, haplotype differences within each group had no visible impact on plasma cystatin C levels which were significantly elevated for all groups compared to the control group. The observed elevation in cystatin C levels was probably a reflection of reduced glomerular filtration rate in these patients. Finally, our results suggest that in this population the haplotype alone was not associated with prognosis and that other, unknown environmental or genetic, factors may be of relevance. Keywords: Cystatin C, promoter, polymorphism, allele, haplotype. Egyptian Journal of Biochemistry and Molecular Biology Vol. 26 (2) 2008: pp. 29-48

A genome-wide association for kidney function and endocrine-related traits in the NHLBI's Framingham Heart Study.

BackgroundGlomerular filtration rate (GFR) and urinary albumin excretion (UAE) are markers of kidney function that are known to be heritable. Many endocrine conditions have strong familial components. We tested for association between the Affymetrix GeneChip Human Mapping 100K single nucleotide polymorphism (SNP) set and measures of kidney function and endocrine traits.MethodsGenotype information on the Affymetrix GeneChip Human Mapping 100K SNP set was available on 1345 participants. Serum creatinine and cystatin-C (cysC; n = 981) were measured at the seventh examination cycle (1998–2001); GFR (n = 1010) was estimated via the Modification of Diet in Renal Disease (MDRD) equation; UAE was measured on spot urine samples during the sixth examination cycle (1995–1998) and was indexed to urinary creatinine (n = 822). Thyroid stimulating hormone (TSH) was measured at the third and fourth examination cycles (1981–1984; 1984–1987) and mean value of the measurements were used (n = 810). Age-sex-adjusted and multivariable-adjusted residuals for these measurements were used in association with genotype data using generalized estimating equations (GEE) and family-based association tests (FBAT) models. We presented the results for association tests using additive allele model. We evaluated associations with 70,987 SNPs on autosomes with minor allele frequencies of at least 0.10, Hardy-Weinberg Equilibrium p-value ≥ 0.001, and call rates of at least 80%.ResultsThe top SNPs associated with these traits using the GEE method were rs2839235 with GFR (p-value 1.6*10-05), rs1158167 with cysC (p-value 8.5*10-09), rs1712790 with UAE (p-value 1.9*10-06), and rs6977660 with TSH (p-value 3.7*10-06), respectively. The top SNPs associated with these traits using the FBAT method were rs6434804 with GFR(p-value 2.4*10-5), rs563754 with cysC (p-value 4.7*10-5), rs1243400 with UAE (p-value 4.8*10-6), and rs4128956 with TSH (p-value 3.6*10-5), respectively. Detailed association test results can be found at . Four SNPs in or near the CST3 gene were highly associated with cysC levels (p-value 8.5*10-09 to 0.007).ConclusionKidney function traits and TSH are associated with SNPs on the Affymetrix GeneChip Human Mapping 100K SNP set. These data will serve as a valuable resource for replication as more SNPs associated with kidney function and endocrine traits are identified.

P1-097: The role of cystatin C in cerebral amyloidosis: New insights from transgenic mouse models

The cysteine protease inhibitor Cystatin C (CysC) is involved in various types of cerebral amyloidosis. The L68Q mutation of CysC causes hereditary cerebral hemorrhages with amyloidosis–Icelandic type (HCHWA–I). Wildtype CysC has been shown to codeposit with Aβ fibrils in the brain of Alzheimer's disease (AD) and HCHWA–Dutch patients. Moreover there is evidence that the B haplotype of the CST3 gene is a risk factor for late onset AD. The aim of this study was to investigate the role of CysC in cerebral amyloidosis in vivo. We generated mouse models overexpressing wildtype or mutated (L68Q) hCysC under the control of a neuron– (Thy1) or an astrocyte–specific (GFAP) promoter. These mice were crossed with APP23 mice (overexpressing hAPP K670N/M671L) or APPPS1 mice (overexpressing hAPP K670N/M671L and hPS1 L166P). Amyloid deposition and protein levels were analyzed by immunohistochemistry and Western blotting. Mice transgenic for mutated CysC, intended to be a model for HCHWA–I, have thus far failed to show any amyloid deposition with aging. However overexpression of wildtype or mutated CysC, either produced by neurons or astrocytes, leads to a reduced Aβ–burden in APP23 and APPPS1 mice. CysC does not seem to affect Aβ–levels in predepositing APP23 mice. Overexpression of wildtype or mutated (L68Q) CysC attenuates Aβ–plaque formation in APP transgenic mice. The observation that CysC produced by astrocytes is able to reduce amyloid formation of neuronally–derived Aβ implies that the interaction of Aβ and CysC occurs extracellularly.

No association between the cystatin C gene polymorphism and Alzheimer's disease: A case-control study in an Italian population

Cystatin C is an amyloidogenic protein found together with beta-amyloid in cerebral arteriolar walls of both patients with Alzheimer's Disease (AD) and conghopilic amyloid angiopathy. Several findings implicate cystatin C in the pathogenesis of vascular diseases. Recent genetic association studies proposed cystatin C gene (CST3) as a susceptibility factor for AD, although other reports did not replicate this finding. We conducted a case-control study including 192 probable AD cases and 192 age- and sex-matched controls to test the association between CST3 and AD. Possible interaction between CST3 and age at onset of AD or apolipoprotein E (APOE) was also examined. No significant differences in CST3 genotype or allele frequencies between cases and controls was observed, while the risk of AD increased in subjects carrying the APOE epsilon4 allele (OR 3.5, 95% CI [2.1-5.9]). There was no interaction between CST3 with age or APOE. Our findings do not support a role of CST3 gene in Italian sporadic AD.

Cystatin C levels are decreased in acute myocardial infarction: Effect of cystatin C G73A gene polymorphism on plasma levels

Background: Cystatin C is the most abundant protease inhibitor in the plasma. Low plasma levels have been found in patients with aortic aneurysms and they seem correlated with the extension of the aortic lesions in early aneurysms detected by ultrasonography. Methods: In this study, plasma levels of cystatin C have been investigated in patients with acute myocardial infarction (AMI), unstable angina and controls. The effect on plasma levels of the G73A polymorphism of the CST3 gene has been also evaluated. Results: Patients with acute myocardial infarction showed significantly lower levels of cystatin C compared to unstable angina and controls, but levels were nearly normal in a week after the acute event. The genotype distribution of the G73A polymorphism was not different among the groups. Nevertheless, cystatin C levels decreased proportionally with the number of A alleles. Cystatin C levels were positively correlated with age, triglyceride/HDL cholesterol ratio and creatinine, and negatively with HDL cholesterol and the number of A alleles. All variables, but not HDL cholesterol, were independently correlated in a multivariate analysis. Conclusions: Cystatin C is decreased in acute myocardial infarction. It is still not clear whether lower cystatin C levels are causally linked to the acute event or just represent a negative acute phase response. The CST3 gene G73A polymorphism functionally affects cystatin C plasma levels.