high-resolution-melting-pcr-for-complement-receptor-1-length

High-resolution Melting PCR for Complement Receptor 1 Length Polymorphism Genotyping: An Innovative Tool for Alzheimer's Disease Gene Susceptibility Assessment

Complement receptor 1 (CR1), a transmembrane glycoprotein that plays a key role in the innate immune system, is expressed on many cell types, but ...

measuring-erythrocyte-complement-receptor-1-using-flow-cytometry

Measuring Erythrocyte Complement Receptor 1 Using Flow Cytometry

CR1 (CD35, Complement Receptor type 1 for C3b/C4b) is a high molecular weight membrane glycoprotein of about 200 kDa that controls complement activation, ...

Reims University Hospitals, Robert Debré Hospital

University of Reims Champagne-Ardenne

Reims University Hospitals, Maison Blanche Hospital

Severe malaria-associated anemia and cerebral malaria are life-threatening complications of Plasmodium falciparum infection. Red blood cell (RBC) complement regulatory proteins (CRPs) have been implicated in the pathogenesis of both. We sought to determine whether there are age-related changes in the expression of CRPs that could explain the susceptibility to severe malaria-associated anemia in young children and the susceptibility to cerebral malaria in older children and adults. In cross-sectional surveys in malaria-endemic and -nonendemic areas of Kenya and in Reims, France, the level of RBC CRPs was lowest in young children and increased into adulthood. In case-control studies, patients with cerebral malaria and matched control subjects had higher levels of RBC CRPs than did patients with severe anemia and matched control subjects, especially during convalescence. We conclude that RBC CRP levels vary with age and that the lower levels of these proteins in young children in areas of high transmission, such as western Kenya, may place these children at greater risk of severe malaria-associated anemia than cerebral malaria.

Age-related changes in red blood cell complement regulatory proteins and susceptibility to severe malaria.

The KN blood group system, which consists of nine antigen specificities, is located on complement receptor Type 1 (CR1/CD35). CR1, a complement regulatory protein, acts as a vehicle for immune complex clearance. CR1 exhibits a red blood cell (RBC) density polymorphism. CR1 sites on RBCs in normal individuals range from 150 to 1200 molecules per cell. CR1 density polymorphism is regulated by HindIII restriction fragment length polymorphism and Q981H and P1786R polymorphisms in Caucasians. Yet, the role of the different polymorphisms in determining the CR1 density on RBCs remains unknown. The "null" serologic KN phenotype, known as Helgeson phenotype, was reported to be related with a very low CR1 density, less than 150 molecules per cell.

Analysis of complement receptor type 1 expression on red blood cells in negative phenotypes of the Knops blood group system, according to CR1 gene allotype polymorphisms.

Common strategy for diagnostics with quantum dots (QDs) utilizes the specificity of monoclonal antibodies (mAbs) for targeting. However QD-mAbs conjugates are not always well-suited for this purpose because of their large size. Here, we engineered ultrasmall nanoprobes through oriented conjugation of QDs with 13-kDa single-domain antibodies (sdAbs) derived from llama IgG. Monomeric sdAbs are 12 times smaller than mAbs and demonstrate excellent capacity for refolding. sdAbs were tagged with QDs through an additional cysteine residue integrated within the C terminal of the sdAb. This approach allowed us to develop sdAbs-QD nanoprobes comprising four copies of sdAbs coupled with a QD in a highly oriented manner. sdAbs-QD conjugates specific to carcinoembryonic antigen (CEA) demonstrated excellent specificity of flow cytometry quantitative discrimination of CEA-positive and CEA-negative tumor cells. Moreover, the immunohistochemical labeling of biopsy samples was found to be comparable or even superior to the quality obtained with gold standard protocols of anatomopathology practice. sdAbs-QD-oriented conjugates as developed represent a new generation of ultrasmall diagnostic probes for applications in high-throughput diagnostic platforms.

Oriented conjugates of single-domain antibodies and quantum dots: toward a new generation of ultrasmall diagnostic nanoprobes.

The long complement receptor type 1 (CR1) isoform, CR1*2 (S), has been identified as being associated with Alzheimer's disease (AD) risk. We aimed to analyze the phenotypic structural and expression aspects (length and density) of CR1 in erythrocytes of 135 Caucasian subjects (100 AD and 35 controls). CR1 length polymorphism was assessed at protein and gene levels using Western blot and high-resolution melting, respectively. CR1 sites on erythrocytes were enumerated by flow cytometry. CR1 gene analysis, spotting the rs6656401 and rs3818361 polymorphisms, was performed by pyrosequencing. The CR1 density was significantly lower in AD patients expressing the CR1*2 isoform compared with the controls (p = 0.001), demonstrating lower expression of CR1 in CR1*2 carriers. Our data suggested the existence of silent CR1 alleles. Finally, rs6656401 and rs3818361 were strongly associated with CR1 length polymorphism (p < 0.0001). These observations indicate that AD susceptibility is associated with the long CR1 isoform (CR1*2), albeit at a lower density, suggesting that AD results from insufficient clearance of plaque deposits rather than increased inflammation.

Alzheimer's disease is associated with low density of the long CR1 isoform.

We developed a two-step PCR-based strategy to detect genes encoding OqxAB, allowing a specific assignment of Tn6010-associated oqxAB in Enterobacteriaceae. Chromosomal location in this setup was confirmed by hybridization with I-CeuI-restricted genomes. This approach led us to find that Klebsiella sp. and Raoultella sp. reference strains chromosomally carried oqxAB.

Discrimination between native and Tn6010-associated oqxAB in Klebsiella spp., Raoultella spp., and other Enterobacteriaceae by using a two-step strategy.

The complement receptor 1 () gene was shown to be involved in Alzheimer's disease (AD). We previously showed that AD is associated with low density of the long CR1 isoform, CR1*2 (S). Here, we correlated phenotype data (CR1 density per erythrocyte (CR1/E), blood soluble CR1 (sCR1)) with genetic data (density/length polymorphisms) in AD patients and healthy controls. CR1/E was enumerated using flow cytometry, while sCR1 was quantified by ELISA.  polymorphisms were assessed using restriction fragment length polymorphism (RFLP), pyrosequencing, and high-resolution melting PCR. In AD patients carrying the H allele (dIII polymorphism) or the Q allele (Q981H polymorphism), CR1/E was significantly lower when compared with controls carrying the same alleles ( < 0.01), contrary to sCR1, which was significantly higher ( < 0.001). Using multivariate analysis, a reduction of 6.68 units in density was associated with an increase of 1% in methylation of  (estimate -6.68; 95% confidence intervals (CIs) -12.37, -0.99;  = 0.02). Our data show that, in addition to inherited genetic factors, low density of CR1/E is also acquired. The involvement of CR1 in the pathogenesis of AD might be linked to insufficient clearance of amyloid deposits. These findings may open perspectives for new therapeutic strategies in AD.

Inherited and Acquired Decrease in Complement Receptor 1 (CR1) Density on Red Blood Cells Associated with High Levels of Soluble CR1 in Alzheimer's Disease.

ArticleTotal : 2	Year
Publication title Cited by 9	2017
Publication title Cited by 3	2020

Article	Year
Age-related changes in red blood cell complement regulatory proteins and susceptibility to severe malaria. The Journal of infectious diseases\| PubMed ID: 15319870	2004
Analysis of complement receptor type 1 expression on red blood cells in negative phenotypes of the Knops blood group system, according to CR1 gene allotype polymorphisms. Transfusion\| PubMed ID: 20210926	2010
Oriented conjugates of single-domain antibodies and quantum dots: toward a new generation of ultrasmall diagnostic nanoprobes. Nanomedicine : nanotechnology, biology, and medicine\| PubMed ID: 21839049	2012
Alzheimer's disease is associated with low density of the long CR1 isoform. Neurobiology of aging\| PubMed ID: 25666996	2015
Discrimination between native and Tn6010-associated oqxAB in Klebsiella spp., Raoultella spp., and other Enterobacteriaceae by using a two-step strategy. Antimicrobial agents and chemotherapy\| PubMed ID: 26124163	2015
Inherited and Acquired Decrease in Complement Receptor 1 (CR1) Density on Red Blood Cells Associated with High Levels of Soluble CR1 in Alzheimer's Disease. International journal of molecular sciences\| PubMed ID: 30044434	2018

Aymric Kisserli

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