We thank all the members of the Plateforme R&#233;gionale de Biologie Innovante, the staff of the Department of Immunology, and the staff of the Department of Internal Medicine and Geriatrics, who contributed to optimizing and validating the protocol. This work was funded by the Reims University Hospitals (grant number AOL11UF9156). We also thank Fiona Ecarnot (EA3920, University Hospital Besancon, France) for editorial assistance.

The protocol for human blood collection and handling was reviewed and approved by the regional ethics committee (CPP Est II), and the protocol number is 2011-A00594-37.
NOTE: The following protocol describes the handling of human blood. Please follow institutional guidelines while disposing of biohazardous material. Laboratory safety equipment, such as lab coats and gloves, should be worn. A flow chart describing the protocol is displayed in Figure 3.
1. Blood and Body Fluid DNA Extraction
<ol>
	<li>Pipet 20 &#181;L of proteinase K (15 &#181;g/&#181;L) into the bottom of a 1.5 mL tube.</li>
	<li>Add 200 &#181;L of sample to the 1.5-mL tube. Use up to 200 &#181;L of whole blood, plasma, serum, buffy coat, or body fluids, or up to 5 x 106 lymphocytes in 200 &#181;L of PBS.</li>
	<li>Add 200 &#181;L of lysis buffer to the sample. Mix by pulse-vortexing for 15 s.</li>
	<li>Incubate at 56 &#176;C for 10 min in a water bath.</li>
	<li>Briefly centrifuge the 1.5 mL tube to remove the drops from the inside of the lid.</li>
	<li>Add 200 &#181;L of ethanol (96-100%) to the sample and mix again by pulse-vortexing for 15 s. After mixing, briefly centrifuge the 1.5 mL tube to remove the drops from the inside of the lid.</li>
	<li>Carefully apply the mixture from step 1.6 to the membrane column (in a 2 mL collection tube). Without wetting the rim, close the cap and centrifuge at 6,000 x g for 1 min. Place the membrane column in a clean 2-mL collection tube and discard the tube containing the filtrate.</li>
	<li>Carefully open the membrane column and add 500 &#181;L of wash buffer 1 without wetting the rim. Close the cap and centrifuge at 6,000 x g for 1 min. Place the membrane column in a clean 2 mL collection tube and discard the tube containing the filtrate.</li>
	<li>Carefully open the membrane column and add 500 &#181;L of wash buffer 2 without wetting the rim. Close the cap and centrifuge at full speed (20,000 x g) for 3 min.</li>
	<li>Place the membrane column in a new 2 mL collection tube and discard the collection tube with the filtrate. Centrifuge at 20,000 x g for 1 min.</li>
	<li>Place the membrane column in a clean 1.5 mL tube and discard the collection tube containing the filtrate.</li>
	<li>Carefully open the membrane column and add 200 &#181;L of distilled water. Incubate at room temperature for 5 min and then centrifuge at 6,000 x g for 1 min.</li>
	<li>Proceed to the determination of DNA concentration step or freeze the DNA samples at -20 &#176;C</li>
</ol>
2. Determination of DNA Concentration
NOTE: See Figure 4.
<ol>
	<li>Press 1 to select DNA mode on a spectrophotometer. Select a 5 mm path length using the left and right arrows. Select a dilution factor of 1 using the left and right arrows.</li>
	<li>Select the unit (&#181;g/mL) using the left and right arrows. Select a factor of 50 using the left and right arrows. Press OK.</li>
	<li>Pipet 10 &#181;L of distilled water into the cuvette. Put the cuvette in the spectrophotometer. Press the OA/100%T button.</li>
	<li>Pipette 10 &#181;L of the DNA sample (1/4 dilution in distilled water) into the cuvette. Put the cuvette in the spectrophotometer. Press the green button (read/start). Note the concentration.</li>
	<li>Freeze the DNA sample at -20 &#176;C.</li>
</ol>
3. HRM-PCR Protocol
<ol>
	<li>Thaw the DNA samples. Dilute the DNA samples in 1.5 mL tubes with water to adjust them to a concentration of 10 ng/&#181;L 
	NOTE: The total volume of diluted DNA should be between 2 &#181;L and 10 &#181;L.</li>
	<li>Thaw the primer solutions. Dilute the primer solutions in 1.5-mL tubes with water to adjust them to the same concentration of 6 &#181;M. 
	NOTE: The primer sequences and reaction conditions are provided in Table 1.</li>
</ol>
<img alt="Table 1" src="/files/ftp_upload/56012/56012table1.jpg" /> 
Table 1: Primers and parameters used in the high-resolution melting analysis.
<ol start="3">
	<li>Thaw the HRM-PCR kit solutions and mix carefully by vortexing to ensure the recovery of all contents. Briefly spin the three vials containing the enzymatic mixture with DNA binding dye, MgCl2, and water in a microcentrifuge before opening them. Store them at room temperature.</li>
	<li>In a 1.5 mL tube at room temperature, prepare the PCR mix for one 20 &#181;L reaction by adding the following components in the order listed below:
	<ol>
		<li>10 &#181;L of enzymatic mixture with DNA binding dye;</li>
		<li>2 &#181;L of 25 mM MgCl2;</li>
		<li>1 &#181;L of primer 1, 6 &#181;M (final concentration: 300 nM);</li>
		<li>1 &#181;L of primer 2, 6 &#181;M (final concentration: 300 nM); and</li>
		<li>5 &#181;L of water. 
		NOTE: To prepare the PCR mix for more than one reaction, multiply the volumes above by the number of reactions to be run, plus one additional reaction.</li>
	</ol>
	</li>
	<li>Mix carefully by vortexing.</li>
	<li>Pipet 19 &#181;L of PCR mix, prepared above, into each well of a white multiwell plate.</li>
	<li>Add 1 &#181;L of concentration-adjusted DNA template, prepared in step 3.1. 
	NOTE: For control reactions, always run a negative control with the samples. To prepare a negative control, replace the template DNA with water.</li>
	<li>Seal the white multiwell plate with sealing foil.</li>
	<li>Place the white multiwell plate in the centrifuge and balance it with a suitable counterweight (i.e., another multiwell plate). Centrifuge for 1 min at 1,500 x g in a standard swing-bucket centrifuge containing a rotor for multiwell plates with suitable adaptors.</li>
	<li>Load the white multiwell plate into the HRM-PCR instrument.</li>
	<li>Start the HRM-PCR program with following PCR conditions: 
	 
	Denaturation: 95 &#176;C for 10 min; 1 cycle. 
	Amplification: 95 &#176;C for 10 s, 62 &#176;C for 15s, and 72 &#176;C for 20s; 47 cycles. 
	Melting curve: 95 &#176;C; ramp rate: 0.02 &#176;C/s; 25 acquisitions per &#176;C; 1 cycle. 
	Cooling: 40 &#176;C for 30 s; ramp rate 2.2 &#176;C/s; 1 cycle.</li>
</ol>
4. HRM Analysis to Determine the CR1 Length Polymorphism
NOTE: The methodology described (Figure 5) is specific to our software (See the Table of Materials), although other software packages may be used.
<ol>
	<li>Open a gene scanning software to perform the CR1 length polymorphism scanning analysis.</li>
	<li>Open the experiment containing the amplification program and the melting curve program.</li>
	<li>Click Sample editor in the Module bar and then select the Scanning workflow.</li>
	<li>Define the properties of the samples (i.e., name; unknown or negative control).</li>
	<li>Click Analysis in the Module bar.</li>
	<li>In the Create New Analysis list, select Gene Scanning.</li>
	<li>Click the Normalization tab to normalize the melting curves.</li>
	<li>Click the Temperature shift tab to reset the temperature axis (x-axis) of the melting curves. 
	NOTE: The lower graph shows melting curves that are both normalized and temperature-shifted.</li>
	<li>Click the Calculate button to analyze the results and determine the grouping.</li>
	<li>Click the Difference plot tab in the charts area to view the Normalized and Shifted Melting Curves and the Normalized and Temperature Shifted Difference Plot.</li>
</ol>

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N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+association+of+CR1+with+Alzheimer's+disease:+a+tentative+disease+mechanism.">Genetic association of CR1 with Alzheimer's disease: a tentative disease mechanism.</a> Neurobiol Aging. 33 (12), 2945-2949 (2012).</li><li>Mahmoudi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease+is+associated+with+low+density+of+the+long+CR1+isoform.">Alzheimer's disease is associated with low density of the long CR1 isoform.</a> Neurobiol. Aging. 36, 1712-1765 (2015).</li><li>Helgeson, M., Swanson, J., Polesky, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knops-Helgeson+(Kna),+a+high-frequency+erythrocyte+antigen.">Knops-Helgeson (Kna), a high-frequency erythrocyte antigen.</a> Transfusion. 10 (3), 137-138 (1970).</li></ol>

The authors are the inventors of a patent owned by the University of Reims Champagne-Ardenne (URCA) (patent number WO 2015166194)

Here, we describe a widely accessible methodology to study CR1 length polymorphisms. The molecular biology techniques for amplification or segmental hybridization have never been able to give satisfactory results that allow for the determination of CR1 length polymorphisms in all individuals. This is because of the repetitive structure of the CR1 gene (i.e., the highly repetitive SCRs of CR1). The molecular biology method described here exploits the quantitative distribution of LHR-B in the CR1 molecule. The CR1 molecule includes n LHR-B domains, where n is a number between 0 and 3, and only one LHR-C domain, as described and illustrated in Figure 2. Quantitative domain B detection makes it possible to perfectly discriminate one additional or missing domain B.
From the extracted genetic material, a first DNA fragment from LHR-B is amplified by PCR using a single pair of specific primers, representing a characteristic variant of LHR-B called &#34;variant amplicon B.&#34; A second DNA fragment, called &#34;invariant amplicon&#34; and belonging to LHR-C, is also amplified. This second DNA fragment is a fragment of LHR-C, but another invariant fragment of LHR-A or -D could also be used.
The two DNA fragments, variant amplicon B and the invariant amplicon, are amplified by the same primers and exhibit five differences in nucleotide sequences. This characteristic makes it possible in the subsequent step to determine the number of variant amplicon B and the number of invariant amplicons using a quantitative molecular biology tool, HRM, which was adapted for this purpose. The ratio between the number of amplicon B and the invariant amplicon (ratio: amplicon B / invariant amplicon) varies according to the length of the CR1 molecule. Indeed, CR1*4 displays 3 amplicon B units to 1 invariant amplicon, CR1*2 displays 2 amplicon B units to 1 invariant amplicon, CR1*1 displays 1 amplicon B to 1 invariant amplicon, and CR1*3 displays 0 amplicon B units to 1 invariant amplicon (Figure 2). Thus, this HRM-PCR method enables the genotyping of the CR1 length polymorphism.
Nevertheless, at the start of the study, this technique requires the use of reference subjects whose CR1 phenotypes are already known (i.e., previously established by WB) in order to be able to establish the genotyping of CR1 according to the reference profile of curves obtained by HRM-PCR. Two types of representation, namely, &#34;Normalized and Shifted Melting Curves&#34; and &#34;Normalized and Temp-Shifted Difference Plot,&#34; are available. They display distinct groups of curve profiles colored according to the CR1 length polymorphism phenotyping obtained by WB (Figure 6). From the &#34;Normalized and Shifted Melting Curves&#34; step, our method makes it possible to discriminate the distinct groups of curves corresponding to the isoforms of CR1, grouped by color. However, the &#34;Normalized and Temp Shifted Difference Plot,&#34; which corresponds to a different mathematical representation, allows for the easier visualization of the distinct groups of curves. Although the manufacturer&#39;s software was routinely used in this study, other software (such as uANALYSE) could be used as a data extraction program for curve analysis.
To date, the WB analysis technique is the original technique that enables the identification of the different CR1 length polymorphisms at the level of the protein. However, this technique has a number of disadvantages. In particular, protein analysis by WB can only be done after the extraction of the cell membranes. As a result, it is complex and very time-consuming. Moreover, this technique requires obtaining a fresh blood sample in appropriate conditions at the beginning of the procedure to achieve useful results. On the contrary, HRM-PCR performed on DNA makes it possible to use even dry blood spots or samples after long-term storage. HMR-PCR requires a specialized DNA melt instrument, either a dedicated instrument or an HMR-capacity thermocycler with HRM software. SNP detection is dependent on several factors: i) SNPs included in large PCR fragments are more difficult to detect than the same SNPs in small PCR fragments; ii) SNPs belonging to classes III and IV are more difficult to detect than those of classes I and II34; and iii) SNPs that are flanked by nearest-neighbor base symmetry (e.g., 5&#39;-G(G/C) C-3&#39;) cannot be sorted by melting, regardless of the resolution power of the HMR platform. It may be useful to test the predicted PCR fragments containing the SNP of interest with the uMELT software35 to assess the validity of the assay. Finally, HMR-PCR is an analogic method, meaning that the similarity of melting curves between a reference and a template does not necessarily imply that the template sequence is identical to the reference, as the template sequence may be different from the reference but thermodynamically equivalent. However, these limitations do not apply to the method described here, which is based on the melting of a mix of amplicons that differ in terms of 5 nucleotide sequences.
In addition, the technique described here, based on the analysis of HRM, has many advantages. First, the CR1 length polymorphisms are determined more rapidly, since the results are obtained in about 2 h once the extraction of the genetic material has been performed. Conversely, traditional biochemical techniques based on WB protein analysis require steps that are relatively long: the electrophoresis of cell membrane extracts through an acrylamide gel, a step to transfer proteins to a nitrocellulose or polyvinylidene fluoride (PVDF) membrane, and a step to identify the isoforms of the CR1 molecule by immunochemiluminescence. Second, the HRM-PCR technique also has the advantage of not being too expensive, which is particularly important for a method likely to be applied to many individuals (i.e., largescale) to determine their susceptibility to the development of pathologies such as Alzheimer&#39;s disease, lupus, or malaria.
Recently, we and others have shown that the CR1*2 isoform, which contains one additional C3b/C4b binding site, was associated with AD36,37,38. In our previously published study, the CR1 length polymorphisms at the level of the gene and the protein were consistent in 98.9% of subjects38. The discordant results obtained when comparing length polymorphisms obtained by HRM-PCR with those obtained by WB corresponded to subjects with a (CR1*1, CR1*2) genotype profile determined by HRM (gene) but expressing only the CR1*1 isoform at the surface of the erythrocyte according to WB (protein). In our study, the lack of CR1*2 isoform expression (individuals bearing a silent allele) was reproducible when the WB was performed with a longer exposure time and could be explained by the existence of a silent CR1 allele (CR1*2), described in the literature by Helgeson39. Nevertheless, further studies on larger populations are needed to support this hypothesis.
It should be noted that private SNPs (SNPs only occurring in a small family group or even in a single individual) led to distinct curve profiles that should be deciphered using reference phenotype determination (WB) but that cannot be confused with the canonical profile to avoid any misinterpretation of the CR1 length polymorphism genotype.

AD, the most common cause of dementia, affects more than 30 million people around the world and is a major public health problem1. Clinically, AD is characterized by neurocognitive disorders leading to a progressive loss of autonomy2. AD is characterized by two neuropathological hallmarks, namely, extra-cellular amyloid deposits and intracellular neurofibrillary tangles3.
Traditionally, according to the age of onset of the disease, AD is classified into two forms. First is early-onset AD (EOAD), where onset most often occurs before the age of 65; this form accounts for less than 5% of AD cases. It is a rare, autosomal-dominant form of AD, which results in fully penetrant mutations either in the amyloid precursor protein (APP)4, presenilin 1 (PSEN1)5, or presenilin 2 (PSEN2)6 genes. Second, the more common form of the disease (&#62;90% of AD cases) is called &#34;sporadic&#34; late-onset AD (LOAD) and most often occurs in individuals aged 65 years or older. It results from multiple genetic and environmental risk factors7. In LOAD, the 4 allele of the apolipoprotein E (APOE) gene is the major genetic risk factor8,9. Furthermore, more than 20 gene loci have been identified by genome-wide association studies (GWAS) as being associated with the risk of AD, one of which being the complement component (3b/4b) receptor 1 (CR1) gene10, located on chromosome 1q32 in a cluster of complement-related proteins. The CR1 gene encodes the complement receptor type 1 (CR1) protein, a component of the complement activity regulators.
CR1 (the C3b/C4b receptor, CD35), a transmembrane glycoprotein of approximately 200 kDa11, binds to the C3b, C4b, C3bi, C1q, mannan-binding lectin (MBL), and ficolin complement proteins12. The biological function of CR1 varies with the cell types in which it is expressed. In humans, 90% of the total circulating CR1 is found in red blood cells (RBCs)13. Present at the surface of RBCs, CR1 binds to C3b- or C4b-opsonized microorganisms or immune complexes, facilitating their clearance from circulation. Complexes bound to CR1 are indeed transferred to phagocytes when RBCs go through the liver and spleen11,14. By limiting the deposition of C3b and C4b, CR1 might prevent excessive complement activation. Therefore, the expression of CR1 on RBCs is considered an essential element in the protection of tissues, such as the cerebral nervous system, against immune complex deposition and the resulting diseases. The CR1 on RBCs is also known to play an important role in pathogenic infection15,16. In addition, CR1, as a key player in innate immunity, is involved in the regulation of the complement cascade and in the transport and clearance of immune complexes. CR1 exerts this activity by binding C3b and C4b fragments and dissociating classical and alternative convertases (dissociation of C2a from the C4b2a complex and dissociation of C3b from the C3bBb complex). As a cofactor of the plasma serine protease factor I (FI), CR1 inhibits the classical and alternative complement pathways by increasing the cleavage of C4b and C3b by FI, a property known as cofactor activity (CA), and by inhibiting the C3 amplification loop, in turn preventing further complement activation. Rogers and colleagues provide evidence that the A&#946; peptide can bind and activate the complement pathway in the absence of antibodies17 and suggest that the A&#946; peptide is cleared from circulation via complement-dependent adherence to the CR1 expressed on RBCs18.
CR1 exhibits three types of polymorphisms: structural or length polymorphisms, density polymorphisms, and Knops blood-group polymorphisms11,19. The structural polymorphism is related to a variation in the number of long homologous repeats (LHRs) and thus defines four isoforms. In fact, the extracellular domain of the CR1 protein is composed of a series of repeating units, called short consensus repeats (SCRs) or complement control repeats (CCPs). These SCRs have been demonstrated from the complement deoxyribonucleic acid (cDNA) encoding CR1. The SCRs are arranged in tandem groups of seven, known as LHRs. CR1 is arranged into four LHRs, designated as LHR-A, -B, -C, and -D, arising from the duplication of a seven-SCR unit19,20,21.
In increasing order of frequency, these CR1 isoforms determined by Western blot (WB) are CR1*1 (A/F) (fast migration on gel electrophoresis), CR1*2 (B/S) (slow migration on gel electrophoresis), CR1*3 (C/F`), and CR1*4 (D). The two most common isoforms, CR1*1 (A/F) and CR1*2 (B/S), are composed of four and five LHRs, respectively, while CR1*3 (C/F`) and CR1*4 (D) are composed of 3 and 6 LHRs, respectively. The most common isoform (CR1*1), composed of 30 SCRs, contains three C4b binding sites (SCRs 1-3; 8-10, and 15-17) and two C3b binding sites (SCRs 8-10 and 15-17), while SCRs 22-28 bind C1q, ficolins, and MBL12,20,21,22,23,24,25. Thus, CR1*2 contains one additional C3b/C4b binding site compared to CR1*1. Figure 1 illustrates the structures, nomenclatures, and molecular weights of the four different isoforms of CR1.
The density polymorphism corresponds to a stable phenotype that represents the level of constitutive expression of CR1 on RBCs. In healthy Caucasian subjects, it has been shown that the number of CR1 molecules per RBC can vary by up to a factor of ten (varying from 150 to 1,200 molecules per cell)26. RBCs of the Helgeson phenotype have a very low CR1 density, which was shown to be lower than 150 molecules per cell27,28. The CR1 density on RBCs is genetically associated with an autosomal codominant biallelic system on the CR1 gene, correlated with a HindIII restriction fragment length polymorphism (RFLP)29. A single-point mutation in Intron 27 of the CR1 gene, between the exons encoding the second SCR in LHR-D, results in the generation of a polymorphic HindIII site within this region30. Genomic HindIII fragments of 7.4 and 6.9 kDa identify alleles associated with high (H allele) or low (L allele) CR1 density on RBCs, respectively. However, no correlation was found between CR1 density on RBCs and HindIII polymorphisms in some West African populations31,32. The mechanism linking CR1 density regulation to a non-coding HindIII polymorphism remains unknown. Among several polymorphisms, Q981H in SCR16 and P1786R in SCR28 have been reported to be linked to the CR1 density on RBCs30,33.
The Knops (KN) polymorphism, according to the international nomenclature, is the 22nd blood group system to be indexed by the International Society of Blood Transfusion. It contains 9 antigenic specificities expressed by the CR1 on RBCs, including three antithetic antigenic pairs, KN1/KN2, KN3/KN6, and KN4/KN7, as well as 3 isolated antigens, KN5, KN8, KN9. The KN1, KN3, KN4, and KN5 antigens are high-frequency antigens of the KN system (i.e., expressed in more than 99% of the general population). However, the role of this polymorphism in AD remains to be determined13.
The protocol described in this work was designed to determine the CR1 length polymorphism genotypes involved in susceptibility to several diseases, such as AD, systemic lupus erythematosus, and malaria. Our method for CR1 length polymorphism determination takes advantage of the number of LHR-Bs comprising the CR1 isoforms and of the sequence differences between LHR-B and LHR-C (Figure 2).

<table><tbody><tr><td>Lab coat</td><td></td><td></td><td>protection</td></tr><tr><td>SensiCareIce powder-free Nitrile Exam gloves</td><td>Medline Industries, Inc, Mundelein, IL 60060, USA</td><td>486802</td><td>sample protection</td></tr><tr><td>Eppendorf Reference 2 pipette, 0,5-10&amp;micro;L</td><td>Eppendorf France SAS, F-78360 Montesson, France</td><td>4920000024</td><td>sample pipetting</td></tr><tr><td>Eppendorf Reference 2 pipette, 20-100&amp;micro;L</td><td>Eppendorf France SAS, F-78360 Montesson, France</td><td>4920000059</td><td>sample pipetting</td></tr><tr><td>Eppendorf Reference 2 pipette, 100-1000&amp;micro;L</td><td>Eppendorf France SAS, F-78360 Montesson, France</td><td>4920000083</td><td>sample pipetting</td></tr><tr><td>TipOne 10&amp;micro;L Graduated, filter tip</td><td>Starlab GmbH, D-22926 Ahrenburg, Germany</td><td>S1121-3810</td><td>sample pipetting</td></tr><tr><td>TipOne 1-100&amp;micro;L bevelled, filter tip</td><td>Starlab GmbH, D-22926 Ahrenburg, Germany</td><td>S1120-1840</td><td>sample pipetting</td></tr><tr><td>ART 1000E Barrier Tip</td><td>Thermo Fischer Scientific , F-67403 Illkirch, France</td><td>2079E</td><td>sample pipetting</td></tr><tr><td>Eppendorf Safe-Lock Tubes, 1.5 mL, Eppendorf Quality</td><td>Eppendorf France SAS, F-78360 Montesson, France</td><td>0030120086</td><td>mix</td></tr><tr><td>Vortex-Genie 2</td><td>Scientific Industries, Inc, Bohemia, NY 111716, USA</td><td>SI-0236</td><td>mix</td></tr><tr><td>QIAamp DNA Mini Kit (250)</td><td>Qiagen SA, F-91974 Courtaboeuf, France</td><td>51306</td><td>DNA Extraction</td></tr><tr><td>Buffer AL</td><td>Qiagen SA, F-91974 Courtaboeuf, France</td><td>19075</td><td>Lysis buffer</td></tr><tr><td>QIAamp Mini Spin Column</td><td>Qiagen SA, F-91974 Courtaboeuf, France</td><td>1011706</td><td>DNA binding</td></tr><tr><td>Buffer AW1</td><td>Qiagen SA, F-91974 Courtaboeuf, France</td><td>19081</td><td>Wash buffer</td></tr><tr><td>Buffer AW2</td><td>Qiagen SA, F-91974 Courtaboeuf, France</td><td>19072</td><td>Wash buffer</td></tr><tr><td>Biowave DNA spectrophotometer</td><td>Biochrom Ltd, Cambridge CB4 OFJ, England</td><td>80-3004-70</td><td>DNA concentration&amp;nbsp;</td></tr><tr><td>Mikro 200 centrifuge</td><td>Hettich Zentrifugen, D-78532, Germany</td><td>0002020-02-00</td><td>centrifugation</td></tr><tr><td>Multipette&amp;nbsp;E3</td><td>Eppendorf France SAS, F-78360 Montesson, France</td><td>4987000010</td><td>distribution</td></tr><tr><td>Light Cycler 480 multiwell plate 96, white</td><td>Roche Diagnostics GmbH, D-68305 Mannheim, Germany</td><td>04729692001</td><td>reaction place</td></tr><tr><td>Light Cycler 480 sealing foil</td><td>Roche Diagnostics GmbH, D-68305 Mannheim, Germany</td><td>04429757001</td><td>coverage</td></tr><tr><td>Heraeus Megafuge 11R centrifuge</td><td>Thermo Fischer Scientific , F-67403 Illkirch, France</td><td>75004412</td><td>centrifugation</td></tr><tr><td>LightCycler 480 Instrument II, 96-well</td><td>Roche Diagnostics GmbH, D-68305 Mannheim, Germany</td><td>05015278001</td><td>high resolution melting polymerase chain reaction</td></tr><tr><td>LightCycler 480 High Resolution Melting Master</td><td>Roche Diagnostics GmbH, D-68305 Mannheim, Germany</td><td>04909631001</td><td>reaction reagents</td></tr><tr><td>light cycler 480 SW 1.5.1 software</td><td>Roche Diagnostics GmbH, D-68305 Mannheim, Germany</td><td></td><td>software used for HRM PCR CR1 polymorphism data analysis</td></tr><tr><td>CN3 primer: 5&#039;ggccttagacttctcctgc 3&#039;</td><td>Eurogentec Biologics Division, B- 4102 Seraing, Belgium</td><td></td><td>reaction reagent</td></tr><tr><td>CN3re primer: 5&#039;gttgacaaattggcggcttcg 3&#039;</td><td>Eurogentec Biologics Division, B- 4102 Seraing, Belgium</td><td></td><td>reaction reagent</td></tr></tbody></table>

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 especially on red blood cells (RBCs). As a receptor for the complement components C3b and C4b, CR1 regulates the activation of the complement cascade and promotes the phagocytosis of immune complexes and cellular debris, as well as the amyloid-beta (A&#946;) peptide in Alzheimer's disease (AD). Several studies have confirmed AD-associated single nucleotide polymorphisms (SNPs), as well as a copy-number variation (CNV) in the CR1 gene. Here, we describe an innovative method for determining the length polymorphism of the CR1 receptor. The receptor includes three domains, called long homologous repeats (LHR)-LHR-A, LHR-C, and LHR-D-and an n domain, LHR-B, where n is an integer between 0 and 3. Using a single pair of specific primers, the genetic material is used to amplify a first fragment of the LHR-B domain (the variant amplicon B) and a second fragment of the LHR-C domain (the invariant amplicon). The variant amplicon B and the invariant amplicon display differences at five nucleotides outside of the hybridization areas of said primers. The numbers of variant amplicons B and of invariant amplicons is deduced using a quantitative tool (high-resolution melting (HRM) curves), and the ratio of the variant amplicon B to the invariant amplicon differs according to the CR1 length polymorphism. This method provides several advantages over the canonical phenotype method, as it does not require fresh material and is cheaper, faster, and therefore applicable to larger populations. Thus, the use of this method should be helpful to better understand the role of CR1 isoforms in the pathogenesis of diseases such as AD.

Figure 6A displays the phenotyping of the CR1 length polymorphism by WB. Figure 6B and C displays the curves that are obtained during HRM-PCR analysis, enabling the determination of the CR1 length polymorphism. Analysis of the fusion curves using the software after the high-resolution melting of the PCR-derived amplicons from the genomic DNA of subjects with different CR1 isoforms produces curves that discriminate subjects according to their allotypic CR1 expression phenotypes.
Figure 6B displays a first mode of presentation, called &#34;Normalized and Shifted Melting Curves,&#34; while Figure 6C displays a second mode of presentation, called the &#34;Normalized and Temp-Shifted Difference Plot.&#34;
The profiles of the curves are distributed according to the groups that represent the phenotypes displayed in Figure 6A: (CR1*3, CR1*1); (CR1*1, CR1*2); CR1*1; CR1*2; and (CR1*2, CR1*4). This enables the genotyping of the CR1 length polymorphism using this new molecular biology method.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56012/56012fig1.jpg" /> 
Figure 1: Schematic representation of the structure and polymorphisms of the complement receptor 1 protein. Each short consensus repeat (SCR) or complement control protein (CCP) is represented by a circle. The SCRs are grouped into a repetitive, higher-order structures called LHRs. From the extracellular domain to the cell membrane, the LHRs are: LHR-A, -B, -C, -D, and -S (supplementary or additional LHR). In the most common CR1 isoform (CR1*1), the C4b/decay accelerating activity (DAA) binding site is located at LHR-A (SCRs 1-3, green circles), the C3b/C4b/cofactor activity (CA) binding site is located in the 3 N-terminal SCRs of LHR-B (SCRs 8-10, red circles) and LHR-C (SCRs 15-17, purple circles), and the binding site for C1q/MBL and ficolin is found in the LHR-D (SCRs 22-28, blue circles). The homologous structures are colored identically. The CR1*2 (S) isoform presents an additional LHR (LHR-S) and thus contains an additional C3b/C4b binding site. KDa, kilodalton. NRC, non-reduced conditions. RC, reduced conditions. TM, transmembrane domain. This figure was adapted from Brouwers et al.36 with permission from Nature Publishing Group. <a href="https://cloudflare.jove.com/files/ftp_upload/56012/56012fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56012/56012fig2.jpg" /> 
Figure 2: Schematic representation of the CR1 isoform structure with sequence amplicon positions obtained by HRM-PCR. Each box represents an SCR. The SCRs are grouped into LHRs: LHR-A, -B, -C and -D. The homologous structures are colored identically. The CR1*2 and CR1*4 isoforms present additional LHRs (LHR-B) and thus contain an additional amplicon area (amplicon B, in red). CR1*3 lacks LHR-B and contains less amplicon area (lacking amplicon B). Nucleotide differences between amplicon B (196 bp) and the invariant amplicon (197 bp) are depicted in red and blue, respectively. Differences in the ratio: (amplicon B, in red / invariant amplicon, in blue) between each CR1 isoform are determined by the HRM-PCR, enabling genotyping. The CN3 and CN3re primer sequences are underlined. TM, transmembrane domain. CYT, cytoplasmic tail. <a href="https://cloudflare.jove.com/files/ftp_upload/56012/56012fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56012/56012fig3.jpg" /> 
Figure 3: Flowchart of the protocol for genotyping the CR1 length polymorphism from human blood samples. Collect a human DNA sample. Extract the DNA to obtain a DNA blood sample. Determine the DNA concentration and dilute to obtain the DNA concentration at 10 ng/&#181;L. Use HRM-PCR to obtain the CR1 length polymorphism genotype. <a href="https://cloudflare.jove.com/files/ftp_upload/56012/56012fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56012/56012fig4.jpg" /> 
Figure 4: Screenshots of the spectrophotometer used to determine the DNA concentration. Press 1 (A) to select DNA mode on the spectrophotometer. Select a 5 mm path length (B) using the left and right arrows (C). Select the unit (&#181;g/mL) (D) using the left and right arrows (C). Select dilution factor 1 (E) using the left and right arrows (C). Select a factor of 50 (F) using the left and right arrows. Press OK (G). Pipet 10 &#181;L of distilled water into the cuvette. Put the cuvette in the spectrophotometer (H). Press the OA/100%T button (I). Pipette 10 &#181;L of DNA sample (1/4 dilution in distilled water) into the cuvette. Put the cuvette in the spectrophotometer (K). Press the green button (read/start) (L). Note the concentration (M). <a href="https://cloudflare.jove.com/files/ftp_upload/56012/56012fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56012/56012fig5.jpg" /> 
Figure 5: Screenshots of the graphical interface of the software used in step 4 of the protocol. (A) Open the gene scanning software. (B) Amplification program and melting curve program. (C) Click on Sample editor in the Module bar. (D) Select Scanning. (E) Define the properties of the samples. (F) Click on Analysis in the Module bar. (G) Click on the Normalization tab to normalize the melting curves. (H) Click on the Temperature shift tab to show the melting curves that are both normalized and temperature-shifted. (I) Click on the Calculate button to analyze the results and determine the grouping. (J) Click on the Difference plot tab to view the Normalized and Shifted Melting Curves and the Normalized and Temperature Shifted Difference Plot. (K) Colored grouping of the samples according to the CR1 length genotypes. <a href="https://cloudflare.jove.com/files/ftp_upload/56012/56012fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56012/56012fig6.jpg" /> 
Figure 6: Phenotyping of the CR1 length polymorphisms observed in the WB and their corresponding profile curves obtained by HRM-PCR. (A) Phenotyping of the CR1 length polymorphisms using WB. (B) and (C) Genotyping of CR1 length polymorphisms using HRM-PCR analysis. HRM curve analysis of PCR amplicons obtained from the genomic DNA of subjects displaying different CR1 isoforms led to the identification of specific curve profiles: (CR1*3, CR1*1) (purple); (CR1*1, CR1*2) (blue); CR1*1 (green); CR1*2 (red); and (CR1*2, CR1*4) (gray). These correspond to the CR1 length polymorphism alleles. As shown by the two modes of presentation-&#34;Normalized and Shifted Melting Curves&#34; (B) and &#34;Normalized and Temp-Shifted Difference Plot&#34; (C)-the curve profiles are distributed according to the (CR1*3, CR1*1); (CR1*1, CR1*2); CR1*1; CR1*2; and (CR1*2, CR1*4) groups. This figure was adapted from Mahmoudi et al.38 with permission from Elsevier. <a href="https://cloudflare.jove.com/files/ftp_upload/56012/56012fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about High-resolution Melting PCR for Complement Receptor 1 Length Polymorphism Genotyping: An Innovative Tool for Alzheimer's Disease Gene Susceptibility Assessmen

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

Here, we describe an innovative method to determine complement receptor 1 (CR1) length polymorphisms for use in several applications, particularly the assessment of susceptibility to diseases such as Alzheimer&#39;s disease (AD). This method could be useful to better understand the role of CR1 isoforms in the pathogenesis of AD.

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

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Research

JoVE Journal

Genetics

11.8K Views.  Reims University Hospitals, Robert Debré Hospital.  Hier beschrijven we een innovatieve methode om complementaire receptor 1 (CR1) lengte polymorfismen te bepalen voor gebruik bij verschillende toepassingen, met name de beoordeling van de gevoeligheid voor ziekten zoals de ziekte van Alzheimer (AD). Deze methode kan nuttig zijn om de rol van CR1-isoformen beter te begrijpen bij de pathogenese van AD. 

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

Selection of Aptamers for Amyloid &beta;-Protein, the Causative Agent of Alzheimer&#39;s Disease

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis difficult1. Definite AD diagnosis is achieved only postmortem, thus establishing presymptomatic, early diagnosis of AD is crucial for developing and administering effective therapies2,3.
Amyloid &beta;-protein (A&beta;) is central to AD pathogenesis. Soluble, oligomeric A&beta; assemblies are believed to affect neurotoxicity underlying synaptic dysfunction and neuron loss in AD4,5. Various forms of soluble A&beta; assemblies have been described, however, their interrelationships and relevance to AD etiology and pathogenesis are complex and not well understood6. Specific molecular recognition tools may unravel the relationships amongst A&beta; assemblies and facilitate detection and characterization of these assemblies early in the disease course before symptoms emerge. Molecular recognition commonly relies on antibodies. However, an alternative class of molecular recognition tools, aptamers, offers important advantages relative to antibodies7,8. Aptamers are oligonucleotides generated by in-vitro selection: systematic evolution of ligands by exponential enrichment (SELEX)9,10. SELEX is an iterative process that, similar to Darwinian evolution, allows selection, amplification, enrichment, and perpetuation of a property, e.g., avid, specific, ligand binding (aptamers) or catalytic activity (ribozymes and DNAzymes).
Despite emergence of aptamers as tools in modern biotechnology and medicine11, they have been underutilized in the amyloid field. Few RNA or ssDNA aptamers have been selected against various forms of prion proteins (PrP)12-16. An RNA aptamer generated against recombinant bovine PrP was shown to recognize bovine PrP-&beta;17, a soluble, oligomeric, &beta;-sheet-rich conformational variant of full-length PrP that forms amyloid fibrils18. Aptamers generated using monomeric and several forms of fibrillar &beta;2-microglobulin (&beta;2m) were found to bind fibrils of certain other amyloidogenic proteins besides &beta;2m fibrils19. Ylera et al. described RNA aptamers selected against immobilized monomeric A&beta;4020. Unexpectedly, these aptamers bound fibrillar A&beta;40. Altogether, these data raise several important questions. Why did aptamers selected against monomeric proteins recognize their polymeric forms? Could aptamers against monomeric and/or oligomeric forms of amyloidogenic proteins be obtained? To address these questions, we attempted to select aptamers for covalently-stabilized oligomeric A&beta;4021 generated using photo-induced cross-linking of unmodified proteins (PICUP)22,23. Similar to previous findings17,19,20, these aptamers reacted with fibrils of A&beta; and several other amyloidogenic proteins likely recognizing a potentially common amyloid structural aptatope21. Here, we present the SELEX methodology used in production of these aptamers21.

Selection of Aptamers for Amyloid &#946;-Protein, the Causative Agent of Alzheimer's Disease

Aptamers are short ribo-/deoxyribo-oligonucleotides selected by in-vitro evolution methods based on affinity for a specific target. Aptamers are molecular recognition tools with versatile therapeutic, diagnostic, and research applications. We demonstrate methods for selection of aptamers for amyloid &beta;-protein, the causative agent of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis ...

Neuroscience

A Method to Study the C924T Polymorphism of the Thromboxane A2 Receptor Gene

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in atherogenesis progression, ischemia, and myocardial infarction. Here we present a methodology of patient genotyping to investigate the post-transcriptional role of the C924T polymorphism (rs4523) situated at the 3&#39; region of the TBXA2 receptor gene. This method relies on DNA extraction from whole blood, polymerase chain reaction (PCR) amplification of the TBXA2 gene portion containing the C924T mutation, and identification of wild type and/or mutant genotypes using a restriction digest analysis, specifically a restriction fragment length polymorphism (RFLP) on agarose gel. In addition, the results were confirmed by sequencing the TBXA2R gene. This method features several potential advantages, such as high efficiency and the rapid identification of the C924T polymorphism by PCR and restriction enzyme analysis. This approach allows a predictive study for plaque formation and atherosclerosis progression by analyzing patient genotypes for the TBXA2R C924T polymorphism. Application of this method has the potential to identify subjects who are more susceptible to atherothrombotic processes, in particular subjects in a high-risk, aspirin-treated group.

In the present study, we describe a methodology to analyze the C924T genotype. The protocol consists of three phases: DNA extraction, amplification by polymerase chain reaction (PCR), and analysis of the restriction fragment length polymorphism (RFLP) on agarose gel.

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in ...

A Robust Polymerase Chain Reaction-based Assay for Quantifying Cytosine-guanine-guanine Trinucleotide Repeats in Fragile X Mental Retardation-1 Gene

Fragile X syndrome (FXS) and associated disorders are caused by expansion of the cytosine-guanine-guanine (CGG) trinucleotide repeat in the 5&#8217; untranslated region (UTR) of the Fragile X mental retardation-1 (FMR1) gene promoter. Conventionally, capillary electrophoresis fragment analysis on a genetic analyzer is used for the sizing of the CGG repeats of FMR1, but additional Southern blot analysis is required for exact measurement when the repeat number is higher than 200. Here, we present an accurate and robust polymerase chain reaction (PCR)-based method for quantification of the CGG repeats of FMR1. The first step of this test is PCR amplification of the repeat sequences in the 5&#8217;UTR of the FMR1 promoter using a Fragile X PCR kit, followed by purification of the PCR products and fragment sizing on a microfluidic capillary electrophoresis instrument, and subsequent interpretation of the number of CGG repeats by referencing standards with known repeats using the analysis software. This PCR-based assay is reproducible and capable of identifying the full range of CGG repeats of FMR1 promoters, including those with a repeat number of more than 200 (classified as full mutation), 55 to 200 (premutation), 46 to 54 (intermediate), and 10 to 45 (normal). It is a cost-effective method that facilitates classification of the FXS and Fragile X-associated disorders with robustness and rapid reporting time.

An accurate and robust polymerase chain reaction-based assay for quantifying cytosine-guanine-guanine trinucleotide repeats in the Fragile X mental retardation-1 gene facilitates molecular diagnosis and screening of Fragile X syndrome and Fragile X-related disorders with shorter turn-around time and investment in equipment.

Fragile X syndrome (FXS) and associated disorders are caused by expansion of the cytosine-guanine-guanine (CGG) trinucleotide repeat in the 5&#8217; ...

Mapping Alzheimer's Disease Variants to Their Target Genes Using Computational Analysis of Chromatin Configuration

Genome-wide association studies (GWAS) have successfully identified hundreds of genomic loci that are associated with human traits and disease. However, because the majority of the genome-wide significant (GWS) loci fall onto the non-coding genome, the functional impact of many remain unknown. Three-dimensional chromatin interactions identified by Hi-C or its derivatives can provide useful tools to annotate these loci by linking non-coding variants to their actionable genes. Here, we outline a protocol to map GWAS non-coding variants to their putative genes using Alzheimer&#39;s disease (AD) GWAS and Hi-C datasets from human adult brain tissue. Putative causal single-nucleotide polymorphisms (SNPs) are identified by application of fine-mapping algorithms. SNPs are then mapped to their putative target genes using enhancer-promoter interactions based on Hi-C. The resulting gene set represents AD risk genes, as they are potentially regulated by AD risk variants. To garner further biological insights into molecular mechanisms underlying AD, we characterize AD risk genes using developmental brain expression data and brain single-cell expression profiles. This protocol can be expanded to any GWAS and Hi-C datasets to identify putative target genes and molecular mechanisms underlying various human traits and diseases.

We present a protocol to identify functional implications of non-coding variants identified by genome-wide association studies (GWAS) using three-dimensional chromatin interactions.

Genome-wide association studies (GWAS) have successfully identified hundreds of genomic loci that are associated with human traits and disease. However, ...

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an upstream 3&#39; splice site within a pre-mRNA, a process called back-splicing. This circularization is likely aided by secondary structures in the pre-mRNA that bring the splice sites into close proximity. In human genes, Alu elements are thought to promote these secondary RNA structures, as Alu elements are abundant and exhibit base complementarities with each other when present in opposite directions in the pre-mRNA. Here, we describe the generation and analysis of large, Alu element containing reporter genes that form circular RNAs. Through optimization of cloning protocols, reporter genes with up to 20 kb insert length can be generated. Their analysis in co-transfection experiments allows the identification of regulatory factors. Thus, this method can identify RNA sequences and cellular components involved in circular RNA formation.

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

We clone and analyze reporter genes generating circular RNAs. These reporter genes are larger than constructs to analyze linear splicing and contain Alu elements. To investigate the circular RNAs, the constructs are transfected into cells and resulting RNA is analyzed using RT-PCR after removal of linear RNA.

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an ...

Biochemistry

Genetic Variant Detection in the CALR gene using High Resolution Melting Analysis

High resolution melting analysis (HRM) is a powerful method for genotyping and genetic variation scanning. Most HRM applications depend on saturating DNA dyes that detect sequence differences, and heteroduplexes that change the shape of the melting curve. Excellent instrument resolution and special data analysis software are needed to identify the small melting curve differences that identify a variant or genotype. Different types of genetic variants with diverse frequencies can be observed in the gene specific for patients with a specific disease, especially cancer and in the CALR gene in patients with Philadelphia chromosome&#8211;negative myeloproliferative neoplasms. Single nucleotide changes, insertions and/or deletions (indels) in the gene of interest can be detected by the HRM analysis. The identification of different types of genetic variants is mostly based on the controls used in the qPCR HRM assay. However, as the product length increases, the difference between wild-type and heterozygote curves becomes smaller, and the type of genetic variant is more difficult to determine. Therefore, where indels are the prevalent genetic variant expected in the gene of interest, an additional method such as agarose gel electrophoresis can be used for the clarification of the HRM result. In some instances, an inconclusive result must be re-checked/re-diagnosed by standard Sanger sequencing. In this retrospective study, we applied the method to JAK2 V617F-negative patients with MPN.

Genetic Variant Detection in the CALR gene using High Resolution Melting Analysis

High resolution melting analysis (HRM) is a sensitive and rapid solution for genetic variant detection. It depends on sequence differences that result in heteroduplexes changing the shape of the melting curve. By combing HRM and agarose gel electrophoresis, different types of genetic variants such as indels can be identified.

High resolution melting analysis (HRM) is a powerful method for genotyping and genetic variation scanning. Most HRM applications depend on saturating DNA ...

Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been accompanied by increased genomic information about Plasmodium parasite species responsible for malaria, including characteristics of geographical populations as well as variations associated with reduced susceptibility to anti-malarial drugs.
One common genetic variation, single-nucleotide polymorphisms (SNPs), offers attractive targets for parasite genotyping. These markers are useful not only for tracking drug resistance markers but also for tracking parasite populations using markers not under drug or other selective pressures.
SNP genotyping methods offer the ability to track drug resistance as well as to fingerprint individual parasites for population surveillance, particularly in response to malaria control efforts in regions nearing elimination status.
While informative SNPs have been identified that are agnostic to specific genotyping technologies, high-resolution melting (HRM) analysis is particularly suited to field-based studies. Compared to standard fluorescent-probe based methods that require individual SNPs in a single labeled probe and offer at best 10% sensitivity to detect SNPs in samples that contain multiple genomes (polygenomic), HRM offers 2-5% sensitivity. Modifications to HRM, such as blocked probes and asymmetric primer concentrations as well as optimization of amplification annealing temperatures to bias PCR towards amplification of the minor allele, further increase the sensitivity of HRM. While the sensitivity improvement depends on the specific assay, we have increased detection sensitivities to less than 1% of the minor allele.
In regions approaching malaria eradication, early detection of emerging or imported drug resistance is essential for prompt response. Similarly, the ability to detect polygenomic infections and differentiate imported parasite types from cryptic local reservoirs can inform control programs.
This manuscript describes modifications to high resolution melting technology that further increase its sensitivity to identify polygenomic infections in patient samples.

While high resolution melting analysis offers the ability to differentiate between single nucleotide polymorphisms in a heterogeneous population, mutant allele amplification bias can increase its ability to detect alleles present at relatively low percentages within a sample. This protocol describes improvements that improve the sensitivity of high resolution melting analysis.

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been ...

Immunology and Infection

Generalized Psychophysiological Interaction (PPI) Analysis of Memory Related Connectivity in Individuals at Genetic Risk for Alzheimer's Disease

In neuroimaging, functional magnetic resonance imaging (fMRI) measures the blood-oxygenation-level dependent (BOLD) signal in the brain. The degree of correlation of the BOLD signal in spatially independent regions of the brain defines the functional connectivity of those regions. During a cognitive fMRI task, a psychophysiological interaction (PPI) analysis can be used to examine changes in the functional connectivity during specific contexts defined by the cognitive task. An example of such a task is one that engages the memory system, asking participants to learn pairs of unrelated words (encoding) and recall the second word in a pair when presented with the first word (retrieval). In the present study, we used this type of associative memory task and a generalized PPI (gPPI) analysis to compare changes in hippocampal connectivity in older adults who are carriers of the Alzheimer's disease (AD) genetic risk factor apolipoprotein-E epsilon-4 (APOE&#949;4). Specifically, we show that the functional connectivity of subregions of the hippocampus changes during encoding and retrieval, the two active phases of the associative memory task. Context-dependent changes in functional connectivity of the hippocampus were significantly different in carriers of APOE&#949;4 compared to non-carriers. PPI analyses make it possible to examine changes in functional connectivity, distinct from univariate main effects, and to compare these changes across groups. Thus, a PPI analysis may reveal complex task effects in specific cohorts that traditional univariate methods do not capture. PPI analyses cannot, however, determine directionality or causality between functionally connected regions. Nevertheless, PPI analyses provide powerful means for generating specific hypotheses regarding functional relationships, which can be tested using causal models. As the brain is increasingly described in terms of connectivity and networks, PPI is an important method for analyzing fMRI task data that is in line with the current conception of the human brain.

Generalized Psychophysiological Interaction PPI Analysis of Memory Related Connectivity in Individuals at Genetic Risk for Alzheimer's Disease

This manuscript describes how to implement a psychophysiological interaction analysis to reveal task-dependent changes in functional connectivity between a selected seed region and voxels in other regions of the brain. Psychophysiological interaction analysis is a popular method to examine task effects on brain connectivity, distinct from traditional univariate activation effects.

In neuroimaging, functional magnetic resonance imaging (fMRI) measures the blood-oxygenation-level dependent (BOLD) signal in the brain. The degree of ...

Genetic Analysis of Hereditary Transthyretin Ala97Ser Related Amyloidosis

Genetic testing is the most reliable test for hereditary transthyretin related amyloidosis and should be performed in most cases of transthyretin amyloidosis (ATTR). ATTR is a rare but fatal disease with heterogeneous phenotypes; therefore, the diagnosis is sometimes delayed. With increasing attention and broader recognition on early manifestations of ATTR as well as emerging treatments, appropriate diagnostic studies, including the transthyretin (TTR) genetic test, to confirm the types and variants of ATTR are therefore fundamental to improve the prognosis. Genetic analyses with polymerase chain reaction (PCR) methods confirm the presence of TTR point mutations much more quickly and safer than conventional methods such as southern blot. Herein, we demonstrate genetic confirmation of the ATTR Ala97Ser mutation, the most common endemic mutation in Taiwan. The protocol comprises four main steps: collecting whole blood specimen, DNA extraction, genetic analysis of all four TTR exons with PCR, and DNA sequencing.

Here, we present a protocol to confirm the presence of point mutation for the diagnosis of hereditary transthyretin amyloidosis, using Ala97Ser, the most common endemic mutation in Taiwan, as an example.

Genetic testing is the most reliable test for hereditary transthyretin related amyloidosis and should be performed in most cases of transthyretin ...

Medicine

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

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

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